Biologically active compounds and methods of constructing and using the same

ABSTRACT

A method of constructing biologically active compounds which mimic the biological activity of the biologically active protein or which block the activity of the biologically active protein is disclosed. A method of identifying specific and discrete portions of pathogen antigens which either serve as epitopes for neutralizing antibodies or which are involved in pathogen binding to host cell receptors is disclosed. A method of constructing biologically active compounds which compete with cellular receptors for binding to either biologically active proteins or pathogen antigens is disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This Application is a Continuation of U.S. patent applicationSer. No. 08/752,816, filed Nov. 21, 1996, allowed, which is aContinuation of U.S. patent application Ser. No. 07/940,654, filed Sep.3, 1992, which issued as U.S. Pat. No. 5,637,677, which is aContinuation-In-Part Application of U.S. patent application Ser. No.07/702,833, filed May 20, 1991, abandoned, which is a ContinuationApplication of U.S. patent application Ser. No. 07/326,328, filed Mar.21, 1989, abandoned, which was a Continuation-In-Part Application ofU.S. patent application Ser. No. 07/074,264, filed Jul. 16, 1987,abandoned. This Application is a Continuation-In-Part Application ofU.S. patent application Ser. No. 07/462,542, filed Jan. 9, 1990,abandoned, which is a Divisional Application of U.S. patent applicationSerial Number 07/074,264, filed Jul. 16, 1987, abandoned. ThisApplication is a Continuation-In-Part Application of U.S. patentapplication Ser. No. 07/648,303, filed Jan. 25, 1991, abandoned, whichis a File Wrapper Continuation Application of U.S. patent applicationSer. No. 07/074,264, filed Jul. 16, 1987, abandoned. This Application isa Continuation-In-Part Application of U.S. patent application Ser. No.07/685,881, filed Apr. 15, 1991, abandoned, which is a ContinuationApplication of U.S. patent application Ser. No. 07/574,391, filed Aug.27, 1990, abandoned which was a File Wrapper Continuation Application ofU.S. patent application Ser. No. 07/194,026 filed May 13, 1988,abandoned, which was a Continuation-In-Part Application of U.S. patentapplication Ser. No. 07/074,264, filed Jul. 16, 1987, abandoned. ThisApplication is a Continuation-In-Part Application of U.S. patentapplication Ser. No. 07/583,626, filed Sep. 14, 1990, abandoned. Each ofthe above listed patent applications is incorporated herein byreference.

ACKNOWLEDGMENT OF GOVERNMENT RIGHTS

[0002] This invention was made with Government support under Grant5R01EY08191 awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to methods of identifying portionsof proteins involved in protein-protein interactions, to methods ofconstructing biologically active peptides involved in protein-proteininteractions, and to biologically active peptides.

BACKGROUND OF THE INVENTION

[0004] Protein binding or protein-protein interactions can be broadlydefined as the discrete interaction of the surface of one protein withthe surface of another protein. Such discrete interaction arises whenresidues of one protein are proximally located to residues of anotherprotein and attractive forces between the residues such as vander Waalsforces, ionic bonds and hydrogen bonds exist. Specific protein-proteininteractions which occur in higher living organisms include but are notlimited to those in which involve: a receptor-binding protein binding toa receptor; a pathogen antigen binding to a host cell receptor; proteininteractions at cellular attachment sites; and, adhesion proteinsinteractions.

[0005] Examples of receptor-binding proteins, hereinafter also referredto as ligands, include cytokines, hormones and growth factors. Theseproteins bind to receptors on cells and cause changes in cellularactivity or function. For example, cytokines are a variety of proteinswhich are cellular messengers, each cytokine having a specific effectupon a cell. Likewise, hormones and growth factors are also messengerswhich affect the function and activity of cells.

[0006] Pathogens are infectious organisms, such as bacteria, fungi,parasites, and viruses and, additionally, neoplasms, all of whichexpress specific antigens. Such typically, there are specific sites onantigens, hereinafter referred to as binding epitopes or epitopes, whichbind to a complementary portion of a cellular protein called a receptorsite.

[0007] A great deal of effort has been expended in search of compoundswhich specifically either simulate, that is mimic, or blockprotein-protein interactions in cells.

[0008] With respect to cytokines, hormones and growth factors, a greatdeal of effort has been made to purify the natural proteins from naturalsources or to synthetically produce them by chemical means or usingrecombinant DNA technology. While some success has been achieved, thesemolecules are quite large, difficult to handle and expensive to obtain.A great deal of effort is also directed at discovering synthetic ligandswhich either mimic the activity of natural proteins or which block theactivity of natural proteins. Blocking natural protein activity can beachieved by either competing for the receptor with an inactive ligand(antagonist) or by having an agent bind to the natural protein andthereby prevent it from binding to the receptor.

[0009] There is a need for synthetic peptides and/proteins which mimicthe activity of the natural biologically active proteins which interactwith receptors. Such mimicking molecules would be useful as agents toaffect the cells in the same way as the natural protein. Likewise, thediscovery of antagonists, that is, molecules which block the receptorwithout having an effect on cellular function or activity would beuseful. Furthermore, the discovery of agents which specifically interactwith biologically active proteins and thereby render them unable to bindto receptors is also desirable. Molecules that prevent binding by anatural biologically active protein to its receptor in cases where thenatural protein is believed to an agent associated with a diseasecondition or disorder are useful as drugs for preventing or treatingsuch disease conditions or disorders.

[0010] A great deal of knowledge has been developed in the field ofimmunology, including at the molecular level. Advances in molecularbiology have indicated that immunoglobulins, major histocompatibilitycomplex antigens and T-cell receptors are all members of a family ofmolecules referred to as the immunoglobulin superfamily. Duringevolution, it is likely that a single, useful gene duplicated, and itscopies diverged to create related molecules with distinct functions.Accordingly, immunoglobulins, which are agents of humoral immunity;T-cell receptors, which are associated with humoral as well as cellularimmunity; and major histocompatibility complex molecules, involved inantigen presentation and the discrimination between self and nonself,all share homologies inherited from their common ancestor and exhibitrelated biological functions.

[0011] Of the members of the superfamily, the structure and function ofimmunoglobulins is best understood. Immunoglobulin molecules consist ofa constant region and a variable region. The constant region isassociated with cellular effector functions whereas the variable regionparticipates in antigen recognition and binding.

[0012] Immunoglobulins of the most common class, IgG, consist of twoheavy chains and two light chains linked together by noncovalentassociations and also by covalent disulfide bonds. Each of the chainspossesses a constant as well as a variable region. In the immunoglobulinmolecule, the variable region is subdivided into framework regions,which are similar in structure among immunoglobulins, and hypervariable,complementarity determining regions (CDRs) which participate directly inantigen binding in the immunoglobulin active site.

[0013] X-ray crystallographic studies of purified immunoglobulinmolecules have indicated that the active site is a crevice formed by theheavy and light chain variable regions, and that the dimensions of theactive sites vary among immunoglobulin molecules consequent to aminoacid sequence variations (Hood et al., 1978, in “Immunology,” TheBenjamin/Cummings publishing Co., Inc., Menlo Park, p. 208). Amino acidsequence, crystallographic structure, and specially designed haptenprobes have been used in conjunction with computer analysis to elucidatethe relationship between an immunoglobulin and the antigen which itrecognizes.

[0014] Pathogens generally express antigens which are recognized by hostimmune systems as foreign and become the target of an immunologicalresponse to eliminate the infectious pathogen. Pathogen antigens oftenbind to cellular receptors on a host's cells as part of the process ofinfection of the host by the pathogen. In order to immunize the host andreduce the effectiveness of the pathogen to mount a challenge to thehost, a number of vaccination strategies have been devised.

[0015] Several strategies have been employed to develop safe, effectivevaccines against viral and bacterial pathogens. At present most vaccinesin use consist of live attenuated pathogens, killed pathogens,components of a pathogen, or modified toxins (toxoids). See Institute ofMedicine, “Vaccine Supply and Innovation”, Washington, D.C.: NationalAcademy Press (1985). While these preparations have been successfullyused for many infectious diseases, many pathogens exist where theseapproaches have not worked or have not been applicable. Certainpathogens are potentially too dangerous to contemplate the use ofattenuated or even inactivated preparations. The risk of developingcancer from immunization with certain retroviruses, or of developingacquired immunodeficiency syndrome (AIDS) from immunization with humanimmunodeficiency virus (HIV) underscores the drawbacks associated withthe use of whole virus preparations for vaccination. In addition manypathogens display a marked antigenic heterogeneity that makes effectivevaccination difficult. These considerations have led us to seekalternative method for effective immunization.

[0016] The idiotype network theory of N.K. Jerne, Ann. Immunol. (Paris)125:337-389, (1974), implies that an anti-idiotypic antibody raisedagainst a neutralizing antibody specific for a pathogen would mimic thatpathogen immunologically. Immunization with the anti-idiotype shouldresult in the development of a significant anti-pathogen response withthe elicitation of neutralizing antibodies and cell-mediated immunity.In recent years there have been several examples where this strategy hasbeen effective, including reovirus type 3. See Sharpe, A. H., et al., J.Exp. Med. 160:195-205 (1984); Kauffman, R. S., et al., J. Immunol.,131:2539-2541, (1983); and Gaulton, G. N., et al., J. Immunol.137:2930-2936. With respect to Sendai virus, see Ertl, H. C. andFinberg, R. W., Proc. Natl. Acad. Sci. USA 81:2850-2854 (1984). Forreport relating to rabies see Reagen, K. J. et al., J. Virol. 48:660-666(1983). This approach has been discussed in connection with polio virusin Uydeltaag, F.G.C.M. and Osterhaus, A.D.M.E., J. Immunol.134:1225-1229 (1985).

[0017] One of the key aspects of this approach is that a portion of theanti-idiotype mimics a portion of the pathogen antigen and induces aneutralizing response. Thus a potent anti-idiotype vaccine would seem tobe an ideal immunogen in cases where intact pathogen could not be usedor where irrelevant non-neutralizing epitopes dominate the immuneresponse. However, the practical application of anti-idiotypes asvaccine has been limited by the difficulties in making human monoclonalantibodies and in the danger of producing serum sickness by usingxenogeneic antibodies.

[0018] Another method currently under intensive investigation is the useof synthetic peptides corresponding to segments of the proteins frompathogenic microorganisms against which an immune response is directed.This approach has been successful in several instances including felineleukemia virus (Elder, J. H. et al., J. Virol. 61:8-15, 1987), hepatitisB (Gerin, J. L., et al., Proc. Natl. Acad. Sci. USA, 80:2365-2369 1983),Plasmodium falciparum (Cheung, A., et al., Proc. Natl. Acad. Sci. USA83:8328-8332, 1986), cholera toxin (Jacob, C. O., et al., Eur. J.Immunol. 16:1057-1062, 1986) and others. When these peptides are capableof eliciting a neutralizing immune response they appear to be idealimmunogens. They elicit a specific response and typically do not lead todeleterious effects on the host. However, it can be difficult to predictwhich peptide fragments will be immunogenic and lead to the developmentof a neutralizing response.

[0019] It would be desirable to develop immunogens that elicit aresponse to specific neutralizing epitopes without causing responses toextraneous epitopes that could “dilute” the specific response or lead toharmful immune complex formation.

[0020] The present invention relates to a method of identifying specificlinear and constrained discrete portions of a biologically activeproteins involved in protein-protein interactions. By identifying suchspecific and discrete portions, biologically active peptides can beconstructed which mimic the biological activity of the biologicallyactive protein or which block the activity of the biologically activeprotein. Thus, biologically active peptides can be constructed which actas ligands that act on mammalian cells by binding to the receptor sitesof those cells to alter or affect their function or behavior, or toprevent the binding of the natural biologically active protein to thecellular receptor, thereby preventing the biologically active proteinfrom affecting the cell.

[0021] The present invention relates to a method of identifying specificlinear and constrained discrete portions of pathogen antigens whicheither serve as epitopes for neutralizing antibodies or which areinvolved in pathogen binding to host cell receptors. By identifyingdiscrete portions of pathogen antigens which are neutralizing epitopes,biologically active peptides can be constructed which are useful ascomponents of vaccines against the pathogen. An effective neutralizingimmune response will be elicited in a vaccinated individual. Byidentifying discrete portions of pathogen antigens which are involved inpathogen binding to host cell receptors, biologically active peptidescan be constructed which are useful as agents which block pathogenattachment to cellular receptors. Additionally, by identifying discreteportions of pathogen antigens which are involved in pathogen binding tohost cell receptors, biologically active peptides can be constructedwhich mimic pathogen antigens and act on mammalian cells by binding tothe receptor sites of those cells to alter or affect their function orbehavior, or which prevent or alter the effect which pathogen antigenswould otherwise have upon those cells.

[0022] The present invention relates to the field of biologically activepeptides which have some shared and/or similar amino acid sequences tothe amino acid sequences of cellular receptor sites and thereby competewith such cellular receptors for binding to either biologically activeproteins or pathogen antigens. In addition, the invention relates to thefield of biologically active peptides which have some shared and/orsimilar amino acid sequences to the amino acid sequences of the ligandsurface that attaches to a cellular receptor site. The ligand mimeticpeptide can be used as a stimulant or inhibitor of that receptor. Wherethe biologically active peptide competes in pathogen/receptor binding,the biologically active peptides are useful to prevent pathogenattachment and thereby prevent infection. Where the biologically activepeptide competes in biologically active protein/receptor binding, thebiologically active peptides are useful to prevent ligand/receptorbinding and thereby prevent the effect on cellular function or behaviornormally associated with the biologically active protein/receptorbinding.

SUMMARY OF THE INVENTION

[0023] One embodiment of the invention relates to a method ofconstructing a peptide capable of eliciting a neutralizing immuneresponse against a pathogen in a mammal. A method of the inventioncomprises the steps of identifying a neutralizing epitope of a pathogenantigen by first generating a neutralizing antibody specific for apathogen, then generating an anti-idiotypic antibody specific for theneutralizing antibody and then identifying the CDR amino acid sequenceof the anti-idiotypic antibody that corresponds to an amino acidsequence of a pathogen antigen. Using that information, a peptide issynthesized that corresponds to or is identical to the portions of theantibody and antigen that correspond to each other.

[0024] Another embodiment of the invention relates to a method ofimmunizing a host mammal against infection by a pathogen that comprisessuch an antigen by inoculating a mammal with a peptide that correspondsto the neutralizing epitope of a pathogen antigen.

[0025] Another embodiment of the invention is a method of constructing apeptide capable of preventing a pathogen or a biologically activeprotein from binding to a cellular receptor. A method of the inventioncomprises identifying an amino acid sequence of a portion of a pathogenantigen or a biologically active protein which binds to the cellularreceptor by first generating an anti-receptor antibody capable ofpreventing a pathogen or a biologically active protein from binding tothe cellular receptor and then identifying an amino acid sequence of theanti-receptor antibody that corresponds to an amino acid sequence of thepathogen antigen or the biologically active protein. Using thatinformation, a peptide is synthesized that corresponds to or isidentical to the portions of the antibody and antigen that correspond toeach other.

[0026] Another embodiment of the invention relates to a method oftreating a host mammal to prevent or reduce the severity of an infectionby a pathogen by constructing a peptide capable of preventing a pathogenfrom binding to cellular receptor and administering the syntheticpeptide to a mammal in an amount effective to prevent or reduce thelikelihood that the pathogen will infect cells of the host.

[0027] Another embodiment of the invention relates to a method ofconstructing a peptide capable of preventing a pathogen or abiologically active protein from binding to a cellular receptor. Amethod of the invention comprises identifying an amino acid sequence ofa cellular receptor which directly interacts with an amino acid sequenceof a pathogen antigen or a biologically active protein during receptorbinding by first generating an antibody specific for the pathogen or thebiologically active protein which is capable of preventing the pathogenor the biologically active protein form binding to the cellular receptorand then identifying an amino acid sequence of the CDR of the antibodywhich corresponds to an amino acid sequence of the cellular receptor.Using that information, a peptide is synthesized that corresponds to oris identical to the portions of the antibody and antigen that correspondto each other.

[0028] Another aspect of the invention relates to a method ofconstructing a biologically active peptide comprising the steps ofidentifying an amino acid sequence of a biologically active portion of abiologically active protein which directly interacts with a cellularreceptor when the biologically active protein binds to the cellularreceptor, wherein such binding causing an effect on an activity orfunction of cell. The amino acid sequence of the biologically activeportion of the biologically active protein identified by firstgenerating an anti-receptor antibody against a cellular receptor, theanti-receptor antibody being capable of effecting an activity orfunction of a cell and then identifying an amino acid sequence of theCDR of the anti-receptor antibody that corresponds to an amino acidsequence of the biologically active protein. Using that information, apeptide is synthesized that is corresponds to or is identical to theportions of the antibody and antigen that correspond to each other.

[0029] Another embodiment of the invention relates to a method ofeffecting or altering activity or function of a mammalian by contactinga cell with an amount of such a biologically active peptide sufficientto effect or alter activity or function of the cell.

[0030] The invention relates to synthetic biologically active peptidescomprising or consisting essentially of amino acid sequence thatcorrespond to an amino acid sequence of an antigen or biologicallyactive protein and an amino acid sequence of an anti-idiotypic antibodyor an anti-receptor antibody.

BRIEF SUMMARY OF THE FIGURES

[0031]FIG. 1 illustrates the specific binding of 9BG5 to peptides,determined by radioimmunoassay as noted in the experimental proceduresdescribed hereinafter; CPM of 9BG5 bound to blank wells was subtractedfrom CPM of 9BG5 bound to peptide coated wells; non-specific binding topeptides was corrected for by subtracting from the value a similar valuedetermined for an isotype-matched control monoclonal antibody UPC10;specific CPM of 9BG5 bound to peptide coated wells is shown using theamount of 9BG5 added to each well in a final volume of 50 μl.; mean±SDfor duplicate wells is shown.

[0032]FIG. 2 illustrates the binding of V_(L)-BSA to type 3 reovirusreceptor as determined by its ability to compete for binding withanti-reovirus type 3 receptor antibody 87.92.6.; R1.1 cells (10⁷/ml)were incubated in 1% BSA in the presence or absence of 200 μg/mlV_(L)-BSA or V_(H)-BSA as indicated for 45 minutes; monoclonalantibodies were added at the concentrations noted for an additional 30minutes; the cells were washed twice and a 1:200 dilution of FITC-goatanti-mouse Fab was added for 30 minutes; the cells were washed twice andanalyzed for fluorescence intensity on a FACS analyzer; percent maximalcell staining was determined as the ratio of the percent of the cellspositive on FACS analysis at the antibody concentration noted to themaximal percent of cells judged positive at saturating doses ofmonoclonal antibody in the absence of competitors ([% positive atconcentration divided by maximal % positive]×100); the maximal percentpositive values were as follows: 2 a-15.3%, 2 b- 97%, 2 c-24%.

[0033]FIG. 3 shows reovirus type 3 and 87.92.6 antibody inhibition of Lcell proliferation.

[0034]FIG. 4 shows inhibition of L cell proliferation by peptides.

[0035]FIG. 5 shows modulation of reovirus type 3 receptor by peptides.

[0036]FIG. 6 shows modulation of the reovirus type 3 receptor bypeptides and antibody.

[0037]FIG. 7 shows inhibition of lymphocyte proliferation.

[0038]FIG. 8 shows peptide inhibition of con A induced lymphocyteproliferation.

[0039]FIG. 9 shows competition of binding of 9BG5 antibody to 87.92.6antibody coated wells in the presence of peptide inhibitors.

[0040]FIG. 10 shows V_(L) and variant peptide inhibition of binding ofreovirus type 3 particles to 9BG5.

[0041]FIG. 11 shows in (a) and (b) V_(L) peptide inhibition of bindingof reovirus type 3 and variant K to L cells; (c) and (d) show V_(L)variant peptide inhibition of binding of reovirus type 3 to murine Lcells.

[0042]FIG. 12 illustrates specific binding of immune serum tovirus-coated plates, determined by radioimmunoassay as noted in thehereinafter described experimental procedures; CPM of immune serumbinding to blank wells was subtracted from CPM binding to virus coatedwells; to account for non-specific binding to virus coated wells, asimilar value determined for normal mouse serum was subtracted form thevalue determined for immune serum; specific CPM bound is shown versusthe dilution of mouse serum added in a final volume of 50 μl.; themean±SEM of duplicate wells from groups of 3 or 4 mice is shown at eachdilution.

[0043]FIG. 13 illustrates immune serum assays for viral neutralizationas described in the following section; serum was collected prior toimmunization with peptides (pre-immune or day 0), on day 20 followingthe first immunization, and on day 60; the neutralization titer wasdetermined at each time point from groups of 4 mice; the geometric meandivided by SEM of the reciprocal of the neutralization titer is shown ateach time point.

[0044]FIG. 14 illustrates plaque inhibition, determined as indicated inthe following description; plaque numbers were determined for 4 mice ineach group and the mean values determined; the highest dilution of serumthat produced 50% or greater plaque inhibition was determined and isshown for each time point at which serum was obtained; plaque inhibitionof both type 1 and type 3 virus is shown.

[0045]FIG. 15 shows the delayed type hypersensitivity (DTH) response ofmice to intact reovirus type 3 after immunization with peptides.

[0046]FIG. 16 shows a representational diagram of two alternate routesfor the development of biologically active peptides according to themethods of the invention.

[0047]FIG. 17 illustrates data for mice immunized with the reovirustypes noted by injection of 10⁷ PFU subcutaneously, or with the peptidesnoted at a dose of 100 μg split into two injections subcutaneously; oneweek later, mice were challenged with virus or peptides in the footpads;footpad swelling was determined as indicated in the followingdescription 48 hours after challenge; the mean±SEM for groups of mice isshown.

[0048]FIG. 18. Structural similarities in gp120 binding domain with Igsuperfamily. Complementarity determining regions (CDR) and frameworkregions (FR) of the first, second, third and fourth domains of therespective heavy (H) or light (L) chains of several antibodies exhibiteda degree of sequence homology with gp120 residues 383-455. The asterisks(*) mark residue positions of shared sequence homology between other HIVisolates and other antibodies. Crystallographic analysis of antibodiesindicates that structural characteristics of CDR regions are preservedin spite of differences in sequence among antibodies. The dash (-) belowa residue position denotes a lack of any sequence homology between anHIV isolated and an antibody. The dash (-) within a sequence denotes adeletion or insertion.

[0049]FIG. 19. Backbone representation of a proposed model for theputative binding side of gp120. The model extends from residue 413through residue 456. The red loop highlights the analogous CDR1-likefold from residues 419-429. The green loop highlights the CDR3L-likedomain from residues 446-456. The disulfide bridge between residues 418and 445 is in yellow. The white loop highlights residues 435-438. Thered and green loops pack in a similar fashion to CDR1L and CDR3L of anantibody molecule with the disulfide positioned in an analogous manner.

[0050]FIG. 20. Comparison of cyclic and linear peptide interactions withthe Reo3R by inhibition of ¹²⁵1-reovirus type 3 binding.

[0051]FIG. 21. Comparison of binding of antisera resulting fromimmunization of rabbits with B138, 466, 1005-45, or 1029-04 peptides togp120.

[0052]FIG. 22. Sequence homology of CD4 and L3T4 with Ig light chains ofknown three-dimensional structure. Boxed areas highlight similarsequences. Dashes (-) indicate insertions/deletions. Sequence alignmentfor comparative model building of CD4 utilizes a crystallographictemplate substituting the sequence of CD4 onto the homologous template.The choice of template is decided based upon the degree of sequencehomology between a template and CD4 and the length of analogousturn/loop structures.

[0053]FIG. 23. Rate of loss of sulfhydryls for various peptides.

[0054]FIG. 24. Binding of 9B.G5 to peptides on solid phase RIA.

[0055]FIG. 25. Inhibition of 9B.G5-87.92.6 interaction by cyclicpeptides.

[0056]FIG. 26. Inhibition of 9B.G5-87.92.6 interaction by cyclicpeptides. Comparison with linear peptides derived from the 87.92.6variable regions.

[0057]FIG. 27. Inhibition of 9B.G5-reovirus type 3 interaction by cyclicpeptides.

[0058]FIG. 28. Inhibition of 9B.G5-reovirus type 3 interaction by cyclicpeptides. Comparison with linear and dimeric peptides derived from the87.92.6 variable regions.

[0059]FIG. 29. Inhibition of 87.92.6-Reo3R interaction by peptides.

[0060]FIG. 30. Specificity of V_(L)C₉C₁₆ peptide binding to the Reo3R.

[0061]FIG. 31. Inhibition of reovirus type 3-Reo3R interaction bypeptides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] According to the invention, specific and discrete portions ofproteins involved in protein-protein interactions can be identified andbiologically active peptides can be constructed based upon the aminoacid sequences identified. The amino acid sequences of specific portionsof anti-idiotypic antibodies correspond to the amino acid sequence ofthe specific portion of the epitope of an antigen that binds to anidiotypic antibody. Likewise, the amino acid sequences of specificportions of anti-receptor antibodies correspond to the amino acidsequences of the specific portion of a ligand which interacts with thereceptor. Thus, the amino acid sequence of either the critical portionof an epitope or the biologically active portion of a biologicallyactive protein can be identified.

[0063] The attachment of proteins to one another often involvessecondary structural features such as loops or helices. The dispositionof specific kinds of residues (aromatic and hydrophilic) allowsattachment to occur through interactions between the residues of thedifferent proteins. These interactions include vander Waals interactionsand hydrogen bonds. The individual loops that occur in portions ofantibodies, for example, form hydrogen bonds with antigen fragments.Likewise, individual loops that occur in portions of receptor moleculesform hydrogen bonds with receptor binding proteins.

[0064] An idiotype is the set of idiotopes which are antigenicdeterminants. The idiotopes occur in the CDR portion of the variableregion of a particular antibody. Antigens represented by an idiotypehave specific interactions with the antibody which results in bind. Suchidiotypes are called internal images of antigens. An anti-idiotypicantibody is an antibody is specific for the portion of another antibodythat represents the idiotope regions. The idiotype or internal image ofan anti-idiotypic antibody is similar to the antigen that the idiotypicantibody recognizes.

[0065] Thus, peptides modelled from the surface of a highly variable CDRloop are used to mimic a region (loop or loop portion of an alpha helix)of some other protein. In some cases, more than one surface can belinked, forming dimers. In other cases, the loops are constrained withspecifically placed cysteine residues or by placement of other residueswhich permit loop closure such as through, for example, ionic bonds.

[0066] As used herein, the term “biologically active protein” refers toproteins which bind to cellular receptors and thereby alter or affectthe function or behavior of the cells, or prevent or alter the effectwhich another biologically active protein would otherwise have uponthose cells. A pathogen antigen can be a biologically active protein if,upon binding to a host cell, it alters or affects the function oractivity of a cell or prevents another agent from doing so. Otherexamples of biologically active proteins include, but are not limitedto, cytokines, hormones and growth factors.

[0067] As used herein, the term “neutralizing epitope” refers to theportion of a pathogen antigen against which antibodies have aneutralizing activity. That is, antibodies specific for a neutralizingepitope will render the pathogen non-infective and/or inactive.

[0068] As used herein, the term “neutralizing antibodies” refers toantibodies which recognize a pathogen and render it non-infective and/orinactive.

[0069] As used herein, the term “anti-pathogen antibodies” refers toantibodies which recognize and bind to a pathogen, specifically apathogen antigen.

[0070] As used herein, the term “anti-receptor antibodies” refers toantibodies which recognize and bind to a receptor, specifically at areceptor site. Anti-receptor antibodies are specific forms ofanti-idiotypic antibodies. Anti-receptor antibodies are anti-idiotypicantibodies which are specific for the idiotype of an immunoglobulinmolecule. That is, they are specific for the portion of theimmunoglobulin receptor which interacts with a biologically activeprotein.

[0071] As used herein, the term “receptor site” refers to the portion ofthe receptor that interacts with a protein that binds to the receptor.

[0072] As used herein, the term “biologically active peptides” refers toproteinaceous molecules which mimic biologically active proteins orprevent the interaction between biologically active proteins andreceptors.

[0073] Biologically active peptides can be constructed which function asthe epitope or mimic a biologically active protein. Alternatively,biologically active peptides can be constructed which interact withreceptors and thereby block the binding of a pathogen antigen orbiologically active protein to a receptor.

[0074] As used herein, the term “biologically active compound” refers toa compound which mimics a biologically active protein or which canotherwise interact with a receptor and thereby block the binding of apathogen antigen or biologically active protein to a receptor.Additionally, a biologically active compound can mimic an epitope of anantigen of a pathogen and elicit a neutralizing immune response in amammal. A biologically active compound may be a peptide or anon-peptidyl compound including, but not limited to, compounds whichcomprise amino acid sequences linked by non-peptide bonds. The term“compounds” as used herein refers to peptides and non-peptidylcompounds.

[0075] One having ordinary skill in the art can appreciate thatbiologically active compounds can be synthesized which comprise aminoacid sequences found in peptides but which are linked by non-peptidebonds. One having ordinary skill in the art can readily appreciate thatthe essential step of identifying the biologically significant portionof an antigen or ligand allows for the construction of compounds,peptide and non-peptide, which mimic the function or activity of theantigen or ligand.

[0076] Accordingly, the methods of the invention also relate toconstructing and using biologically active compounds that are modelledbased upon corresponding amino acid sequences of antigen or ligands andanti-idiotypic or anti-receptor antibodies. The identification ofcorresponding sequences in portions of anti-idiotypic antibodies oranti-receptor antibodies and pathogen antigens or biologically activeproteins can be used in the construction of biologically activecompounds which comprise such shared amino acid sequences but which arelinked by non-peptide bonds. Furthermore, using well known techniques,such non-peptide biologically active compounds can be synthesized fromreadily available starting materials be those having ordinary skill inthe art.

[0077] As used herein, the terms “correspond” and “corresponding” referto the level of shared identity between two amino acid sequences. Thatis, the amount of identical and conservatively substituted amino acidsequences shared between two molecules. As used herein, two sequencescorrespond if, when compared, they share approximately at least 80%identical and conservatively substituted sequences of which at leastabout 28% are identical sequences and between about 30-42% conservativesubstitutions. Generally, corresponding amino acid sequences share atleast six similar amino acid residues. Corresponding sequences are oftenlonger, comprising about 10 or more corresponding residues. As usedherein, these terms refer to the quantifiable similarity between aminoacid sequences. One having ordinary skill in the art can compare aminoacid sequences and calculate whether or not they correspond to eachother. The terms “homologous”, “homology”, and “sequence similarity” areoften used interchangably by those having ordinary skill in the art torefer to corresponding amino acid sequences.

[0078] One having ordinary skill in the art can determine that an aminoacid sequence corresponds to another amino acid sequences. The level ofskill of those having ordinary skill in the art provides that amino acidsequences can be compared and sequence “similarity”, “homology”, and“correspondence” can be determined routinely. The processes of comparingand determining sequences correspondence are well known and widelyreported. See, for example, Bruck, C. et al., 1986 Proc. Natl. Acad.Sci. USA, 83:6578-6582, which is incorporated herein by reference. Onehaving ordinary skill in the art can construct a peptide having an aminoacid sequence which corresponds to another amino acid sequence.Corresponding amino acid sequences can be determined and peptides can beconstructed using other amino acid sequences as models. The amino acidsequence of such a peptide can be identical to that sequence from whichit was modelled. Peptides can be constructed that comprise amino acidsequences modelled after two corresponding sequences. An amino acidsequence can be determined which corresponds to both model sequences.

[0079] When the anti-idiotypic antibody is specific for an anti-receptorantibody, the specific portion of the receptor involved inligand/receptor interaction or pathogen/receptor interaction can beidentified. Peptides can be constructed which bind to the ligand orpathogen at the specific portion normally involved in receptor binding,thereby preventing receptor binding.

[0080] Harlow, E. and D. Lane, ANTIBODIES: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor N.Y. 1988, which isincorporated herein by reference, provide a review of the molecular andgenetic aspects of mammalian immunology generally, and antibodies inparticular. This publication contains a review of antibody structure andfunction including variable regions and the CDRs thereof.

[0081] Antibodies bind to antigens by virtue of their secondarystructure. Antibodies contains amino acid sequences within the CDR ofthe variable region that form loops or reverse turns. The CDRs representdeterminants termed idiotypes. CDRs are quite variable in sequence.However, their shapes are limited. Therefore, the number ofconformations of the CDRs are limited. The peculiar shape orconformation of the CDRs is determined by a few amino acid residues.Therefore, one can imagine antibodies of a certain specificity as havinga conserved beta sheet framework and loop projections which aregenerally comparable. The individual amino acid residues on each loopmight be different when one compares one antibody to another. Theconsequence is that a particular loops which bind antigens may vary insequence from one antibody to another but will often resemble each otherin three dimensional structure. Since amino acid sequences of antibodiesin their CDRs are hypervariable, they themselves can resemble foreignantigens which have similar loops containing similar amino acids.Accordingly, the means by which antibodies bind other proteins can beapplied to construct immunogens or mimetics of immunogens orbiologically active proteins.

[0082] It is well known to those having ordinary skill in the art thatthe light and heavy chains of antibodies contain variable regions and,within these regions, three loop portions known as CDRs which arehypervariable. The CDRs are the portions of the antibodies where bindingto epitopes takes place. That is, CDRs of an antibody comprise aminoacid sequences which form a three dimensional structure that directlyinteracts with the three dimensional structure formed by specific aminoacid sequences of the antigen to which the antibodies bind. A specificportion of the CDR loop interacts with a specific portion of an antigenmolecule. Methods of determining the amino acid sequence of the variableregions of antibodies are well known to those having ordinary skill inthe art.

[0083] According to the invention, identification of the amino acidsequence of the portion of antibody that reacts to a specific portion ofthe target protein can be used to construct biologically activecompounds. It has been discovered that anti-idiotypic antibodies containregions of peptide sequences which correspond to the peptide sequencesof the epitope for which the anti-pathogen antibody binds. It has beendiscovered that anti-receptor antibodies contain regions of peptidesequences which correspond to the peptide sequences of pathogen antigensor natural biologically active proteins which bind to such receptors.

[0084] Amino acid sequences of anti-idiotypic antibodies andanti-receptor antibodies which correspond to and which mimic epitopesand biologically active portions of biologically active proteins,respectively, it has been discovered that such corresponding sequencesoccur in the variable regions of the antibodies. The correspondingregions usually occur within the CDRs, often within the CDR II, that isthe designated second CDR. In particular, the corresponding sequencesusually occur within one or two of the CDR of the light chain and/orheavy chain. In the case of anti-idiotypic antibodies, it has beendiscovered that portions of those antibodies which specifically interactwith antibodies against an antigen correspond to portions of theantigen. Similarly, in the case of anti-receptor antibodies, it has beendiscovered that they correspond to, that is, they share sequencesimilarity with, portions of pathogen antigens associated with pathogenattachment to cellular receptors and with portions of biologicallyactive proteins that interact with receptors.

[0085] This technology is particularly useful to identify amino acidsequences for the following purposes.

[0086] 1. To construct immunogenic compounds which mimic pathogenantigen neutralizing epitopes and are thereby useful to elicitneutralizing antibodies against pathogens, such compounds are useful asvaccine components.

[0087] 2. To construct biologically active compounds which block thebinding of pathogens to receptors on host cells and thereby preventpathogen attachment which is usually essential in pathogen infection. Inorder to prevent pathogen/receptor interaction, such compounds caneither bind to the pathogen antigen that binds to the receptor or to thereceptor.

[0088] 3. To construct biologically active compounds which mimicbiologically active proteins by binding to the receptor sites of thosecells, such binding causing alterations or effects to cellular functionor behavior. Examples of such biologically active proteins includecytokines, hormones and growth factors.

[0089] 4. To construct biologically active compounds which bind to thereceptor sites of those cells to prevent or alter the effect which abiologically active proteins would otherwise have upon those cells.

[0090] 5. To construct biologically active compounds which bind tobiologically active proteins, preventing the biologically active proteinfrom binding to the receptor site on a cell, thereby preventing theprotein from causing the effect which a biologically active proteinwould otherwise have upon those cell.

[0091] The invention can be practiced by modeling compounds based uponsimilarity between pathogen antigens or biologically active proteins andthe CDR loops on the loop portions of the alpha helix of ananti-idiotypic antibodies or by modeling peptides based upon similaritybetween pathogen antigens or biologically active proteins andanti-receptor antibodies. In each case, the same result is achieved.That is, the identification of portions of an antibody sequence, a CDRloop, which are identical or similar to relevant significant regions ofa biologically significant protein; i.e. epitopes of antigens orbiologically active portions of biologically active proteins.

[0092] In the case of construction of immunogenic compounds which mimicpathogen antigen neutralizing epitopes, anti-idiotypic antibodiesspecific for antibodies against the pathogen antigen neutralizingepitope contain sequences corresponding to the pathogen antigenneutralizing epitope. The pathogen antigen neutralizing epitope can beidentified by comparing the amino acid sequence of the pathogen antigento the amino acid sequence of the anti-idiotypic antibodies,particularly the variable regions, particularly the CDR regions. Byidentifying which portion of the pathogen antigen contains theneutralizing epitope, compounds such as peptides can be synthesizedwhich are either identical or similar to the epitope of the antigen orto the region of the antibody. Vaccines can be formulated which includesuch compounds. These compounds will elicit a neutralizing antibodyresponse and immunity or protection from pathogenic infection will beconferred upon the subject of the vaccination.

[0093] In the case of construction of biologically active peptides whichblock the binding of pathogens to receptors on host cells, the aminoacid sequences of pathogen antigens involved in such binding can beidentified by raising antibodies against the receptor and comparing theamino acid sequence of the pathogen antigen to the amino acid sequenceof the anti-receptor antibodies, particularly the variable regions,particularly the CDR.

[0094] Alternatively, anti-idiotypic antibodies specific for antibodiesthat bind to pathogen antigens and thereby prevent binding of thepathogen to the receptor can contain amino acid sequences thatcorrespond to the amino acid sequences of the pathogen antigen whichbinds to the receptor. The amino acid sequence of the pathogen antigenthat binds to the receptor can be identified by comparing the amino acidsequence of the pathogen antigen to the amino acid sequence of theanti-idiotypic antibodies, particularly the variable regions,particularly the CDR regions.

[0095] In either of these cases, by identifying the portion of thepathogen antigen that binds to the receptor, compounds can besynthesized which are either identical or similar to the antigensequence or to the region of the antibody. The peptides can beadministered to a patient. These compounds will block a pathogen frombinding to the receptor and thereby prevent pathogen attachment which isusually essential in pathogen infection.

[0096] Pathogen binding to cellular receptors has been associated withalterations or effects on cell function and activity. In order toconstruct biologically active peptides which mimic the binding ofpathogens to receptors on host cells, the amino acid sequences ofpathogen antigens involved in such activity can be identified by raisingantibodies against the receptor which mimic the activity and comparingthe amino acid sequence of the pathogen antigen to the amino acidsequence of the anti-receptor antibodies, particularly the variableregions, particularly the CDR regions. Compounds can be constructedwhich are based upon the portions of both molecules that correspond toeach other, that is, that share sequence similarity. Such compounds willeither block the mimic the effect that pathogen binding has on cells orprevent pathogen binding from occurring and thereby prevent the effectscaused by pathogen binding.

[0097] It is contemplated that pathogen/receptor binding can beprevented by constructing biologically active compounds which mimic thereceptor site and bind to the pathogen antigen. Such compounds areessentially “caps” to the antigen's receptor binding site and preventthe antigen from interacting with the receptor. In order to constructsuch biologically active compounds, the amino acid sequences of receptorsite involved in pathogen binding can be identified by raisinganti-idiotypic antibodies specific for anti-receptor antibodies thatblock pathogen binding and comparing the amino acid sequence of thereceptor to the amino acid sequence of the anti-idiotypic antibodies,particularly the variable regions, particularly the CDR regions.Compounds can be constructed which are based upon the portions of bothmolecules that correspond to each other, that is, that share sequencesimilarity. Such compounds will mimic the receptor site and bind to thepathogen antigen at the receptor binding site, preventing the pathogenfrom binding to the receptor.

[0098] In another embodiment of the invention, biologically activecompounds can be constructed by identifying the biologically activeportion of a biologically active protein. The biologically activeportion of a biologically active protein can be identified by generatingantibodies specific for the receptor with which the biologically activeprotein interacts. Such antibodies must either block the binding of thebiologically active protein of the receptor or mimic the activity of thebiologically active protein. The amino acid sequence of the biologicallyactive protein is compared to the amino acid sequence of theanti-receptor antibodies, particularly the variable regions,particularly the CDR regions. Compounds can be constructed which arebased upon the corresponding portions of both molecules, that is, thatportions that share sequence similarity. Such compounds will eitherblock the receptor or mimic the activity of the biologically activeprotein.

[0099] In another embodiment of the invention, binding of a biologicallyactive protein to a receptor can be prevented by constructingbiologically active compounds which mimic the receptor site and bind tothe biologically active portion of the biologically active protein. Suchcompounds are essentially “caps” to the biologically active protein'sreceptor binding site and prevent the biologically active protein frominteracting with the receptor. In order to construct such biologicallyactive compound, the amino acid sequences of receptor site involved inbiologically active protein/receptor binding can be identified byraising anti-idiotypic antibodies specific for anti-receptor antibodiesthat block biologically active proteins from binding to the receptor andcomparing the amino acid sequence of the receptor to the amino acidsequence of the anti-idiotypic antibodies, particularly the variableregions, particularly the CDR regions. Compounds can be constructedwhich are based upon the corresponding portions of both molecules, thatis, the portions that share sequence similarity. Such compounds willmimic the receptor site and bind to the biologically active protein atthe receptor binding site, preventing the biologically active proteinfrom binding to the receptor and thereby neutralizing its ability toaffect cells.

[0100] The essence of the invention is the discovery that the specificportion of anti-idiotypic antibody or an anti-receptor antibody thatrecognizes a neutralizing antibody or a receptor, respectively,corresponds to the neutralizing epitope of an antigen or thebiologically active portion of a biologically active protein whichnormally binds to the receptor, respectively.

[0101] The techniques needed to practice the invention are well known tothose having ordinary skill in the art. The starting materials needed topractice the invention are readily available.

[0102] Antibodies against a pathogen, a receptor or another antibody areproduced by routine methods. One having ordinary skill in the art candesign assays to determine whether an antibody is a neutralizingantibody. Such assays are well known and their design and operationroutine. Similarly, one having ordinary skill in the art can designassays to detect whether a pathogen is blocked from attaching to acellular receptor. Such assays are well known and their design andoperation routine. Furthermore, one having ordinary skill in the art candesign assays to determine the biological activity of a peptideincluding its ability to block the activity of another molecule are wellknown. Such assays are well known and their design and operationroutine.

[0103] Amino acid sequence determination can be readily accomplished bythose having ordinary skill in the art using well known techniques.Generally, DNA sequencing of relevant genetic material can be performedand the amino acid sequence can be predicted from that information.Sequencing of genetic material, including the variable regions ofantibodies, particularly the CDRs, can be performed by routine methodsby those having ordinary skill in the art.

[0104] One having ordinary skill in the art can readily determinewhether or not one amino acid sequence corresponds to another. Thedetermination of whether sequences are corresponding may be based on acomparison of amino acid or nucleic acid sequence, and/or proteinstructure, between the protein of interest, that is, the pathogenantigen, cellular receptor or biologically active protein, and a memberof the immunoglobulin superfamily, in particular anti-idiotypicantibodies or anti-receptor antibodies, particularly the CDRs of thevariable regions of such antibodies.

[0105] By determining the number of identical and conservativelysubstituted amino acid sequences shared between two molecules, onehaving ordinary skill in the art can determine whether or not twosequences correspond. The two sequences correspond if they shareapproximately at least 80% identical and conservatively substitutedsequences of which at least about 28% are identical sequences andbetween about 30-42% conservative substitutions. Generally,corresponding amino acid sequences share at least six similar amino acidresidues. Corresponding sequences are often longer, comprising about 10or more similar residues. One having ordinary skill in the art, usingroutine techniques can by quantification determine whether two sequencesare correspond within the meaning used herein.

[0106] Assays to determine whether or not antibodies are useful in amethod to identify biologically active peptides can be readily designedand performed by those having ordinary skill in the art. Determinationof whether an anti-pathogen antibody is neutralizing can be done bythose having ordinary skill in the art. Determination of whether ananti-receptor antibody mimics or blocks a biologically active proteincan be done by those having ordinary skill in the art.

[0107] Antibodies are generated against a pathogen by routine methodsand, if they are found to be neutralizing, that is, if they preventinfection, anti-idiotypic antibodies are generated against theanti-pathogen antibodies. If the anti-idiotypic antibodies are capableof eliciting neutralizing antibodies, the anti-idiotypic antibodies aresequenced. Sequencing of the antibody can be directed at the variableregions, particularly the CDRs, by well known methods. The portion ofthe amino acid sequence of the antibody that corresponds to an aminoacid sequence of the antigen of the pathogen is identified by sequencingboth the antibody and the pathogen. The portion of the antibody wherethe similarity usually occurs is the variable region, in particular theCDR. A peptide is constructed which contains the amino acid sequence ofthe pathogen that corresponds to a portion of the anti-idiotypicantibody or which contains the amino acid sequence of the correspondingportion of the anti-idiotypic antibody. The peptide's ability to elicita neutralizing antibody is confirmed. The peptide is useful in a vaccineto protect against infection of the host by the pathogen.

[0108] Antibodies are generated against a receptor that a pathogen bindsto in order to attach to a cell. An assay can be performed to determinewhether or not the anti-receptor antibody prevents the pathogen frombinding to the receptor. The portion of the antibody corresponding tothe antigen involved in receptor binding is identified by sequencing theantibody. Sequencing of the antibody can be directed at the variableregions, particularly the CDRs, by well known methods. The peptide issynthesized and will block prevent pathogen attachment to the receptor.The peptide is formulated as a pharmaceutic which is administered, forexample, as a therapeutic to combat pathogen infection.

[0109] Pathogens and biologically active proteins such as cytokine,hormones and growth factors, bind to cellular receptors and alter theactivity or function of a cell. Biologically active peptides areconstructed according to the invention which, by binding to thereceptor, mimic the effect that pathogens or biologically activeproteins have on cells. Alternatively, biologically active peptides areconstructed which prevent the binding of pathogens or biologicallyactive proteins to the receptor and thereby prevent or alter the effectthose agents would otherwise have upon the cells.

[0110] Antibodies are generated against a receptor and selected fortheir ability to mimic the effect that pathogens or biologically activeproteins have on cells. If the antibodies are active, the portion of theantibody that is corresponds to either a portion of the pathogen antigeninvolved in receptor binding or a portion of the biologically activeprotein is identified by sequencing the antibody and the pathogenantigen or biologically active protein, respectively. Sequencing of theantibody can be directed at the variable regions, particularly the CDRs,by well known methods. The peptide is synthesized and will mimic thepathogen or biologically active protein. The peptide is formulated as apharmaceutic which is administered, for example, as a therapeutic toelicit the activity of that the native proteins have on cells.

[0111] In order to identify biologically active peptides which preventbiologically active proteins from binding to cellular receptors,antibodies are generated against the receptors. Antibodies that competewith biologically active proteins in binding to the receptor but that donot mimic the effect that biologically active proteins have on cells areselected. If the antibodies are block binding but are not active, theportion of the antibody that corresponds to a portion of thebiologically active protein is identified by sequencing the antibody andbiologically active protein. Sequencing of the antibody can be directedat the variable regions, particularly the CDRs, by well known methods.The peptide is synthesized and will block the biologically activeprotein but will not mimic its activity. The peptide is formulated as apharmaceutic which is administered, for example, as a therapeutic tocounteract the activity of the biologically active protein.

[0112] Biologically active compounds, such as peptides, can beconstructed which mimic the binding site of the receptor and therebybind to the binding portion of either a pathogen antigen or abiologically active protein. Such peptides bind to the pathogen antigenor biologically active protein, effectively preventing those proteinsfrom binding to the receptor. In order to identify biologically activepeptides which mimic receptor binding sites and bind to either pathogenantigens or biologically active proteins, antibodies are generatedagainst the pathogen antigens or biologically active proteins receptors.Alternatively, anti-idiotypic antibodies raised against anti-receptorantibodies can also be used. The antibodies are tested to identify thosethat prevent pathogen antigens or biologically active proteins frombinding to cellular receptors. Antibodies that compete with receptors tobind with pathogen antigens or biologically active proteins areselected. If the antibodies are block binding, the portion of theantibody that corresponds to a portion of the receptor is identified bysequencing the antibody and receptor. Sequencing of the antibody can bedirected at the variable regions, particularly the CDRs, by well knownmethods. The peptide is synthesized and will bind to either the pathogenantigen or the biologically active protein, thus preventing thoseproteins from binding to the receptors. The peptide is formulated as apharmaceutic which is administered, for example, as a therapeutic tocounteract the activity of the biologically active protein.

[0113] Peptides can be synthesized by those having ordinary skill in theart using well known techniques and readily available startingmaterials. According to the invention, references to synthesizing orconstructing peptides is herein construed to refer to the production ofpeptides similar in sequence or structure to the corresponding regionsidentified by the method of the invention. These peptides may beproduced using any method known in the art, including, but not limitedto, chemical synthesis as well as biological synthesis in an in vitro orin vivo in a eukaryotic or prokaryotic expression system. The peptidesmay consist of only corresponding regions or they may comprise thecorresponding sequences and addition sequences.

[0114] Peptides of the invention may be biologically active as producedor may require modification in order to assume a three-dimensionalconformation which is biologically active. Generally, the peptides areactive as produced. However, some modifications may be necessary foractivity and some modifications may be desirable to improve or alteractivity.

[0115] Modifications which may be performed, using standard techniques,according to the invention include but are not limited to cyclization,disulfide bond formation, glycosylation, phosphorylation, or theaddition or subtraction of amino acid residues including amino acidresidues which serve to produce a useful three dimensional conformationvia a chemical linkage which is not generally found in natural peptidesand/or mimetics including but not limited to, those described inFreidinger et al., 1980, Science 210:656; Hinds et al., 1988, J. Chem.Soc. Chem. Comm. 1447; Kemp et al., 1984, J. Org. Chem. 49:2286; Kemp etal., 1985, J. Org. Chem. 50:5834; Kemp et al., 1988, Tetrahedron Lett.29:5077; Jones et al., 1988, Tetrahedron Lett. 29:3853.

[0116] Additionally, modifications may be performed, using standardtechniques, according to the invention to create dimers or oligomers ofthe loops or multi-looped structures.

[0117] An increase or decrease in bioactivity associated withmodification may be ascertained using the appropriate assay system. Forexample, if the activity of the peptide is associated withimmunogenicity, the ability of modified and unmodified peptides toelicit an immune response may be compared.

[0118] Further, if the desired geometry of a peptide is known, computermodelling may be used to identify modifications of the peptide whichwould result in the desired geometry. The success of these modificationsin increasing bioactivity could then be evaluated using in vitro or invivo assay systems.

EXAMPLES Example 1

[0119] The following embodiments of the invention are described inconnection with experiments which have been conducted using reovirustypes 1 and 3 interactions with cellular receptors using theanti-idiotype anti-receptor approach.

MATERIALS AND METHODS

[0120] Mice

[0121] Adult Balb/c female mice, 6 to 8 weeks to age, were obtained fromJackson Laboratories, Bar Harbor, Me. Pre-immune serum was obtained onall mice used and assayed by neutralization of reovirus infectivity (seebelow) to ascertain that there had been no prior exposure to reovirus.Mice immunized with peptides were housed in the animal care facility andfed a house diet ad libitum (Purina, St. Louis, Mo.). Mice immunizedwith reovirus type 3/Dearing were housed in a separate facility.

[0122] Viruses

[0123] Reovirus type 1 (Lang), and reovirus type 3 (Dearing) and thereassortants 3.HA-1 and 1.HA-3 have been previously described (Fields,B. N. and Greene, M. I., Nature 20:19-23, 1982) . Clones 1.HA-3 and3.HA-1 are single segment reassortant clones that segregate the S1 gene,the gene encoding the viral attachment polypeptide (hemagglutinin)sigma 1. For mouse inoculation and virus neutralization, a stock ofreovirus that was passed twice in L-cells was purified by substitutingultrasonic disruption (Branson Ultrasonic 200) for cell homogenizationin a modification of published techniques (Joklik, W. K., Virology49:700-715, 1972). The number of particles per ml was determined byoptical density at 260 nm (Smith, R. E. et al., Virology 39:791-810,1969).

[0124] Monoclonal Antibodies

[0125] Type 3 reovirus neutralizing monoclonal antibody 9BG5 (mouseIgG2aK) (Burstin, S. J. et al., Virology 117:146-155, 1982) was purifiedfrom hybridoma supernatant with the cells grown in Dulbecco's minimalessential media (DMEM) (MA Bioproducts, Walkersville, Md.) with addedpenicillin/streptomycin solution (The Cell Center, University ofPennsylvania, Philadelphia, Pa.), and 10% fetal bovine serum (FBS).Culture supernatants were precipitated with 50% (NH₄)₂SO₄, solubilizedin distilled water and dialyzed against three changes of phosphatebuffered saline (PBS). Next, the antibody was purified on aSepharose-protein A column and eluted with 0.1 M citric acid pH 4.5. Theeluate was collected in 1 M tris buffer, pH 8.5 to neutralize excessacidity and dialyzed against three changes of PBS. The dialysate wasconcentrated on an Amicon protein concentrator with a molecular weightcut-off of 30 kilodaltons (kD). The purified antibody was more than 95%pure by sodium-dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Irrelevant monoclonal antibodies UPC-10 and All (both mouseIgG2aK) were similarly purified from clarified ascites (Gibco, GrandIsland Biological Co.).

[0126] Monoclonal antibodies 87.92.6 (mouse IgM, K) and HO 13.4 (mouseIgM, K, anti-Thy 1.2) and HO 22.1 (mouse IgM, K, anti-Thy 1.1) werepurified from 50% ammonium sulfate cuts of culture supernatant or fromascites supernatants from ascites generated in hybridoma bearing Balb/cmice. These preparations were dialyzed against three changes of PBS andrun over a goat anti-mouse IgM Affigel-10 column. Antibodies were elutedwith 3.5 M MgCl₂, dialyzed against three changes of PBS and concentratedas noted above. Purity of all monoclonal antibodies used was greaterthan 95% by SDS-PAGE.

[0127] Cell Lines

[0128] Murine L-cells were grown in spinner bottles with Joklik's MEM(MA Bioproducts) with 5% FBS. R1.1 cells (murine thymoma, Thy 1.2+) weregrown in suspension in RPMI 1640 (MA Bioproducts, Walkersville, Md.)supplemented with L-glutamine, 10 mM HEPES buffer (MA Bioproducts) andpenicillin/streptomycin with 10% FBS.

[0129] Immunization of Mice

[0130] For the study of DTH response, groups of mice were inoculatedwith either synthetic peptide or live reovirus type 3 subcutaneously(s.c.) in two separate sites on the dorsal flanks of the animal (overeach hind limb); 50 μg of a synthetic peptide or 10⁷ viral particles/0.2ml were given in separate injections of 0.1 ml vol. Six days later,animals were challenged in the left footpad with 3×10⁷ viral particlessuspended in saline containing 2% gelatin (30 μl). Footpad swelling wasrecorded 24 hr later in a blind fashion (Greene, M. I. and Weiner, H.L., J. Immunol. 125:283-287, 1980). Four animals per group were studied,and the magnitude of the response was determined by comparing thechallenged left footpad to the untreated right footpad.

[0131] For the study of humoral immune response, mice were inoculatedwith either synthetic peptide or live reovirus type 3 as above with thefollowing modification. The peptide was conjugated with chicken serumalbumin (CSA) as described below and 100 μg of the peptide conjugate wasinoculated s.c. in two divided doses. For mice immunized with syntheticpeptides, the first immunization was with peptide mixed with an equalvolume of complete Freund's adjuvant; whereas with subsequentimmunization the peptide was suspended in saline containing gelatin.Mice were immunized weekly for five weeks, and serum was obtained priorto the first inoculation, and then at the second and sixth week. Formice immunized with reovirus type 3, 10⁷ plaque forming units (PFU) wasinoculated s.c. on the first and third week.

[0132] Radioimmunoassav Procedure

[0133] The wells of 96 well V-bottom polystyrene plates (DynatechLaboratories, Alexandria, Va.) were coated with peptide by diluting thepeptides to 25 μg/ml in distilled water and evaporating 50 μl in eachwell by incubating the plates overnight at 37° C. Wells were coated withreovirus type 1 or type 3 by diluting stock solutions of virus to4.8×10¹¹ particles/ml in 0.1 M NaHCO₃ pH 9.5, dispensing 25 μl per welland incubation overnight at 4° C. (London, S. D., et al. 1987).Following overnight incubation, peptide or virus coated wells werewashed three times with PBS and blocked with 200 μl/well of 1% gelatinin PBS with 0.1% NaHCO₃ by incubation for 2 hours at 37° C. The wellswere decanted, washed three times in PBS, and mouse serum or purifiedmonoclonal antibody was added, 50 μl/well, diluted in PBS containing0.5% gelatin and 0.1% NaN₃. Following a 3 hour incubation at 37° C., thewells were decanted, washed three times in PBS, and radioiodinated goatanti-mouse Kappa diluted in PBS 0.1% NaHCO₃ with 1 mg/ml chicken gammaglobulin was added, 100 μl=48,000 counts per minute (CPM) per well. Theplates were incubated overnight at 4° C., decanted, washed ten times intap water and dried under a heat lamp. Wells were then cut out using ahot wire and counted in a gamma counter. The CPM determined on blankwells not coated with antigen is subtracted from CPM values determinedon antigen coated wells in all cases.

[0134] Fluorescence Activated Cell Sorter (FACS) Analysis

[0135] R1.1 cells (99% viability to trypan blue dye exclusion) werecentrifuged and washed twice in PBS 0.1% NaN₃ with 1% bovine serumalbumin (FACS media). Cells were resuspended at 10⁷/ml either in FACSmedia alone or FACS media containing peptide-BSA conjugates at 200μg/ml. The cells were incubated on ice for 45 minutes prior to additionof monoclonal antibodies from 0.5 mg/ml stock solutions to 100 Alaliquots to the final concentrations noted. Following an additional 30minute incubation, 500 μl of FACS medium was added to each sample, thecells were centrifuged, washed once in 500 μl FACS media, resuspended in10 μl FACS containing a 1:200 dilution of fluoresceinated goatanti-mouse Fab (Southern Biotechnology Associates) and incubated for 30minutes on ice. 500 μl of FACS media was added, the cells werecentrifuged and washed in 500 μl FACS media, resuspended in 200 μl FACSmedia and analyzed at the University of Pennsylvania fluorescenceactivated cell sorter.

[0136] Neutralization of Virus Infectivity

[0137] The titer of neutralizing antibodies in serum sample weredetermined in the following manner:

[0138] (i) Micro-neutralization: L-cells (5×10⁴ per well) were incubatedin 96 well dishes overnight at 37° C. Reovirus type 1/Lang (1/L) andtype 3/D were serially diluted and incubated for 1 hour with the L-cellsat 37° C. An additional 75 μl of MEM supplemented with 5% fetal bovineserum, 1% glutamine was placed in each well. At 3 days followingincubation at 37°, the media containing virus was removed and the cellswere stained with Gentian Violet (Gentian Violet 3.4 g/l, ammoniumoxalate 8 g/l). The titer of virus used for neutralization was 4 fold inexcess of that quantity of virus that was lytic for the L-cellmonolayer. Reovirus type 1/L or 3/D at the appropriate concentration wasincubated with an equal volume of mouse serum for 1 hr at 25° C. on 96well plates. The virus-serum mixture was then transferred to L-cellmonolayers as above. The titer of antibody was determined as the amountwhich preserved 70% of the monolayer as determined by visual inspection.

[0139] (ii) Virus plaque reduction: 100 pfu of reovirus type 1/Lincubated for 1 hour with L-cells (7×10⁵ cells per well) in 12 wellCostar plates. The titer of virus in each well was then determined aspreviously described (Rubin, D. H., J. Virol. 53:391-398, 1985).

[0140] Synthesis of Peptides

[0141] Peptides were synthesized using a model 430A Applied BiosystemsPeptide Synthesizer (Applied Biosystems, Inc., Foster City, Calif.).Deprotection and release of the peptide from the solid phase supportmatrix were accomplished by treating the protected peptide on the resinwith anhydrous HF containing 10% anisole or 10% thioanisole for 1 to 2hr at 0° C. The peptides were then extracted with either ethyl acetateor diethylether and then dissolved in 10% aqueous acetic acid andfiltered to remove the resin. After lyophilization, the composition andpurity of the peptides were determined with both amino acid analysis andreverse phase high performance liquid chromatography. This procedure wasused for the synthesis of all peptides, including V_(L) and the variantpeptides of V_(L).

[0142] Conjugation of Peptides to Chicken Serum Albumin (CSA)

[0143] Prior to conjugating the peptides to CSA, the CSA was firstderivatized with a nucleophilic spacer consisting of adipic dihydrazide,as described by Schneerson, et al., J. Exp. Med. 152:361, (1980). 30 mgof the adipic dihydrazide-derivatized-CSA (CSA-ADH) in 5 ml 0.1M sodiumbicarbonate was reacted for 15 min at room temperature with 7 mgm-maleimidobenzoylsulfosuccinimide ester (Pierce). To this reactionmixture was then added 50 mg peptide and the couples reaction was allowsto proceed at 25° C. for 2 hr. Following dialysis against 0.1M ammoniumbicarbonate and lyophilization, the CSA-ADH-peptide conjugates wereobtained as dry white powders.

RESULTS

[0144] Determination of Shared Peptide Sequence

[0145] Prior work has shown that a monoclonal antibody denoted 87.92.6raised against monoclonal neutralizing anti-reovirus antibody 9BG5mimics the intact virus by binding to cell-surface receptors specificfor type 3 reovirus. See Noseworthy, J. H. et al., J. Immunol.131:2533-2538, 1983; Kauffman, R. S., et al, 1983 supra; and Co, M. S.et al., Proc. Natl. Acad, Sci. USA 82:1494-1498, 1985. Monoclonalantibody 87.92.6 competes with reovirus type 3 for binding to specificcellular receptors thereby mimicking the viral cell attachment proteinsigma 1 (the viral hemagglutinin) in its binding domain. This domain isalso implicated in the neutralizing antibody response (Burstin, S. J.,et al, 1982 supra; Spriggs, D. R. et al., Virology 127:220-224 1983).This implies that 87.92.6 mimics the epitope on the hemagglutinin thatinteracts with the cellular receptor for reovirus.

[0146] The nucleic acid sequences of the heavy and light chain variableregions (V_(H) and V_(L) respectively) of 87.92.6 have recently beendetermined (Bruck, C. et al., Proc. Natl. Acad. Sci. USA 83:6578-6582,1986), and the sequences have been compared to that of the reovirus type3 sigma 1 protein (Bassel-Duby, R. et al., Nature 315:421-423, 1985). Inaccordance with the methods of the invention, shared sequence portionsof the antigen and anti-idiotype have been identified. Moreparticularly, a 16 amino acid sequence in the reovirus type 3 sigma 1protein encompassing amino acids 317 and 332 has been identified ashaving corresponding amino acid sequences to a combined sequenceencompassing the second complementarity determining regions (CDR II's)of the 87.92.6 heavy and light chain variable regions (V_(H) and V_(L)respectively). Specifically, amino acids 43-51 of the V_(H) sharesequence similarity with amino acids 317-324 of sigma 1 and amino acids46-55 of the V_(L) correspond to amino acids 323-332 of sigma 1 (Bruck,C., et al, 1986, supra).

[0147] In accordance with the methods of the invention, peptidescorresponding to amino acids 317-332 of the sigma 1 protein 43-50 of theV_(H) sequence and 39-55 of the V_(L) sequence have been synthesized. Asdemonstrated hereinafter, immunization of Balb/c mice with thesepeptides results in neutralizing anti-reovirus type 3 antibodies andspecific cell-mediated immunity to reovirus. This establishes that thecorresponding sequences between the sigma 1 cell attachment protein andthe anti-receptor antibody predicts the neutralizing epitope on thereovirus hemagglutinin, sigma 1. This approach allows the rapiddelineation of neutralizing epitopes on pathogens and the development ofpeptide vaccines that elicit a neutralizing response.

[0148] Binding of Neutralizing Monoclonal Antibody 9BG5 to Peptides

[0149] The monoclonal anti-receptor antibody 87.92.6 binds to both thereovirus type 3 receptor and the neutralizing antibody 9BG5 (Kauffman,R. S., et al, 1983, supra) . Applicants predicted that the peptidesderived from the areas of similarity between 87.92.6 and the type 3reovirus sigma 1 protein (Bruck, C., et al, 1986 supra) would havesimilar properties. The peptides synthesized to test this hypothesis areshown in Table I.

[0150] The peptides used in this study were synthesized by solid-phasemethods as noted above. The sequences are shown aligned with maximumsimilarity. The amino acids marked with a closed circle are identicaland those marked with an open circle are of the same class. It will benoted that the tested peptides contain anti-idiotypic antibody residuesin addition to the shared peptide sequence.

[0151] The reo peptide corresponds to amino acids 317-332 in the type 3viral hemagglutinin. Computer modeling predicts this area to bepredominantly a beta-sheet configuration and to include a beta-turn. TheV_(L) peptide represents amino acids 39-55 of the light chain variableregion of 87.92.6, and includes the second complementarity determiningregion (CDR II). Modeling predicts this area also to be a predominantbeta-sheet and to include a beta-turn. The V_(H) peptide comprises aminoacids 43-56 of the heavy chain variable region of 87.92.6 including CDRII of the heavy chain. The control peptide, unrelated to this system, isalso shown.

[0152] Based on these similarities in primary and secondary structures,it was predicted that the reo and V_(L) peptides should be recognized byanti-reovirus type 3 neutralizing monoclonal antibody 9BG5. FIG. 1 showsthe results of a radioimmunoassay determining the binding of purifiedmonoclonal antibody 9BG5 to the wells of microtiter plates coated withthe peptides. To control for non-specific binding to the polystyrenewells, counts per minute (CPM) determined on blank wells not coated withpeptide is subtracted from CPM values determined on peptide coatedwells. In addition, since these peptides may also cause non-specificadherence of immunoglobulin molecules, the specific binding of theclass-matched irrelevant monoclonal antibody UPC-10 to peptide coatedwells and subtracted this value from those determined for 9BG5 wasdetermined. No significant binding was seen to the control peptide usedin this study. Similarly, binding to the V_(H) peptide only achievedbackground levels indicating that this epitope is not recognized by9BG5. There was a small amount of binding to the V_(L) peptide, whichhas strong similarity in its carboxy terminal sequence to the reopeptide carboxy terminal. Although slight, this finding was reproducibleon subsequent assays. Strong reproducible binding to the reo peptide by9BG5 was evident. Since 9BG5 is a neutralizing antibody, this datumimplies that the reo peptide contains the neutralizing epitoperecognized by 9BG5. The binding to the V_(L) peptide indicates that thearea of sequence between these peptides (amino acids 323-332 of thesigma 1 protein) is involved in the neutralizing epitope.

[0153] Binding of V_(L) Peptide to the Reovirus Receptor

[0154] Prior work indicated that the neutralizing epitope recognized by9BG5 is involved in binding to the type 3 reovirus receptor (Kauffman,R. S., et al. (1983) supra; Noseworthy, J. H., et al. (1983) supra;Spriggs, D. R., et al. (1983) supra) . It was therefore speculated thatthe V_(L) peptide might also interact with the viral receptor. To testthis hypothesis the V_(H) and V_(L) peptides were coupled to BSA byincubating peptides and BSA in 0.1% glutaraldehyde followed by dialysisagainst PBS. These preparations were used to determine if 87.92.6specifically blocked binding to the type 3 reovirus receptor on R1.1cells. As shown in FIG. 2a, pre-incubation of R1.1 cells with V_(L)-BSAblocked the binding of 87.92.6 indicating interaction of V_(L)-BSA withthe reovirus receptor. This blocking effect is specific aspre-incubation of R1.1 cells with V_(L)-BSA had no effect on the bindingof HO 13.4, and isotype matched control monoclonal antibody that bindsto the Thy 1.2 molecule on the R1.1 cell surface (FIG. 2b). Theseobservations were consistently reproducible on multiple experiments. Anadditional control is shown in FIG. 2c where it is demonstrated thatV_(H)-BSA has no inhibitory effect on 87.92.6 binding when used at thesame concentrations as V_(L)-BSA. These data indicate a directinteraction of the V_(L) peptide with the reovirus type 3 receptor andimply that residues 46-55 of the 87.92.6 V_(L) chain and 323-332 of thetype 3 sigma 1 protein directly interact with the reovirus type 3receptor.

[0155] Binding of Reovirus type 3 Inhibits Host Cell DNA Synthesis UponReceptor Perturbation

[0156] Reovirus type 3 inhibits host cell DNA synthesis upon receptorperturbation. This effect is not due to infection of cells asreplication defective reovirus type 3 particles retain this property. Lcells were cultured at 5×10⁴ cells per well of 96 well microtiter platesin 100 μl media for 24 hours. Reovirus type 3 particles (A) were addedand incubated for an additional 24 hours prior to the addition oftritiated thymidine. Purified monoclonal antibodies 87.92.6 or HO 22.1(B) were added for 1 hour at 37° C., then the culture media removed andreplaced with 100 μl fresh media for 24 hours, prior to the addition oftritiated thymidine. The cells were incubated for an additional 4-6hours and counts per minute (CPM) incorporated were determined.

[0157]FIG. 3 shows this effect of reovirus type 3 upon murinefibroblasts. Murine fibroblasts (which posses specific receptors forreovirus type 3) (L cells), were incubated with reovirus type 3, or leftuntreated (3A). Twenty-four hours later the DNA synthetic level wasmeasured. Reovirus type 3 markedly inhibited DNA syntheses by thesecells. 87.92.6 has a similar effect on these cells, as shown in FIG. 3B.In this experiment, L cells were grown adherent and exposed to antibodyfor one hour, at which point the antibody was removed, and the cellscultured for an additional 24 hours prior to determination of the DNAsynthesis while a control antibody (H022.1) had no effect. 87.92.6similarly inhibits DNA synthesis by fibroblasts, neuronal cells andlymphocytes.

[0158] Binding of Dimeric Peptides to Reovirus Type 3 Receptors

[0159] It was reasoned that V_(L) peptide may exhibit biologic effectssimilar to those exhibited by reovirus type 3 and 87.92.6. 87.92.6 iseffective only as a native antibody while monomeric Fab fragments haveno effect. V_(L) peptide was synthesized with an additional aminoterminal cysteine residue (V_(L)SH) to form a dimeric peptide. V_(L)SHpeptide was dimerized by stirring a 5 mg/ml solution in 0.1 M ammoniumbicarbonate overnight at 23° C. exposed to air. The peptides were thenlyophilized. Dimerization was confirmed by Ellman determinationaccording to the procedure of Ellman, G. L. Arch. Biochem. Biophys.74:443 (1958), which revealed less than 5% free sulfhydryl groups. LCells were suspended at 10⁶ cells/ml in DMED 10% FBS and 50 μl added toeach well of 96 well microtiter plates. Following 24 hours of culture,peptides were added to the concentrations noted, and the cells culturedfor an additional 24 hours. Tritiated thymidine was added for anadditional 4-6 hours, and counts per minute (CPM) incorporated wasdetermined. Per cent inhibition was determined by the formula:$\left\lbrack {1 - \frac{\left( {{CPM}\quad {without}\quad {additive}} \right) - \left( {{CPM}\quad {with}\quad {additive}} \right.}{{CPM}\quad {without}\quad {additive}}} \right\rbrack \times 100$

[0160] The peptides utilized were: V_(L):Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser- Gly-Ser-Thr-Leu-GlnV_(L)SH: Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln Control:Cys-Tyr-Thr-Tyr-Pro-Lys-Glu-Asp-Thr-Ala-Asn-Asn- Arg

[0161] As shown in FIG. 4, marked inhibition of DNA synthesis wasobserved when L cells were treated with V_(L)SH. V_(L) peptide monomers(without the added cysteine residue) had no effect on L cellproliferation. Several control peptides utilized also had no effect inthese assays (FIG. 4). This indicates that aggregation of the reovirustype 3 receptor on L cells is essential for the inhibition of DNAsynthesis by these peptides.

[0162] Down-Modulation of Reovirus Type 3 Receptor By Peptide Dimers

[0163] Aggregation of the reovirus type 3 receptor on some cells by87.92.6 leads to disappearance of that receptor from the cell surface.It was reasoned that V_(L)SH peptide might similarly down-modulate thisreceptor. For these experiments we utilized murine thymoma (R1.1) cells,which have well characterized reovirus type 3 receptors were utilized.The effect of peptides on the level of expression of both the reovirustype 3 receptor (recognized by 87.92.6) and Thy 1.2 molecules(recognized by HO 13.4), as determined by flow cytometry was studied.R1.1 cells were cultured with peptides at the concentration noted (A),left untreated (B), or treated with 500 μg/ml peptide (C,D) for 1 hourat 37° C. The cells were centrifuged and washed three times in 1% BSA inPBS with 0.1% sodium azide (FACS media). Monoclonal antibodies 87.92.6(100 μl of affinity purified antibody) was added for 30 minutes on ice.The cells were washed and 100 μl of a 1:100 dilution of fluoresceinatedgoat anti-mouse Ig (Southern Biotechnology Associates, Birmingham, Al.)was added for 30 minutes. The cells were washed and fluorescenceintensity analyzed by flow cytometry. Mean channel fluorescence wascompared for cells incubated in the presence or absence of primaryantibody to give mean channel fluorescence (FIG. 4, panel A). Cells werestained with HO 13.4 (FIG. 4, left panels in A, B, C, and D) which bindsThy 1.2 molecules, or with 87.92.6 (FIG. 5, right panels in A, B, C, andD) which binds the reovirus type 3 receptor. Cells were treated withV_(H) peptide (FIG. 5, panel A, left, and panel C) or V_(L)SH peptide(FIG. 5, panel A, right, and panel D).

[0164] The V_(H) peptide sequence:

V_(H):Cys—Gln—Gly—Leu—Glu—Gln—Ile—Gly—Arg—Ile—Pro—Ala—Asn—Gly

[0165] The other peptides are those described above for FIG. 4. As shownin FIG. 4, V_(L)SH peptide specifically down-modulates the reovirus type3 receptor in a dose-dependent manner, but does not effect theexpression of Thy 1.2 molecules on these cells. This down-modulation isa direct biologic effect of V_(L)SH peptide and not due to other factorsin the experimental design. The control peptide used (V_(H) peptide)does not effect the level of expression of the reovirus type 3 receptor,or of Thy 1.2 molecules, on these cells. V_(H) peptide was derived fromthe 87.92.6 heavy chain CDR II and does not specifically interact withthe reovirus type 3 receptor. It has been demonstrated previously thatV_(L) peptide in this form does not compete with 87.92.6 for binding tothese cells, although other forms of V_(L) peptide are able to inhibit87.92.6 binding. In addition, in the studies described in FIG. 5, thecells were washed thoroughly to remove free V_(L)SH peptides prior toflow cytometry. Collectively these data indicate that competition forbinding to the reovirus type 3 receptor is not responsible for thedecreased staining with 87.92.6. The down-modulation of the reovirustype 3 receptor accounts for this phenomenon.

[0166] Receptor down-modulation is dependent on aggregation of thereceptor, as demonstrated in FIG. 6. Data from three experimentscomparing the effect of V_(L) peptide monomers and V_(L)SH peptide isshown. R1.1 cells were treated as described above with peptides (100μg/ml) or 87.92.6 (a 1:1 dilution of ascites), and analyzed forexpression of the reovirus type 3 receptor (87.92.6) or Thy 1.2molecules (HO 13.4). Per cent decrease in mean channel fluorescence iscalculated as follows: The mean channel fluorescence of peptide orantibody treated cells is subtracted from that of untreated cells, thisdivided by the mean channel fluorescence of untreated cells; theresultant value is subtracted for 1 and multiplied by 100. For peptidetreated cells, mean channel fluorescence is determined on peptidetreated cells in the presence or absence of primary antibody. Forantibody treated cells, mean channel fluorescence is determined by themean channel number of antibody treated cells in the presence of primaryantibody minus the mean channel number of untreated cells in the absenceof primary antibody. Cells treated with antibody and then analyzedwithout primary antibody staining had an increase in mean channel numberwhen compared with untreated cells. The mean±standard deviation from 3experiments is shown for peptide treated cells. The peptides used inthese experiments included: V_(L):Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser- Gly-Ser-Thr-Leu-GlnV_(L)SH: Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln Control:Cys-Thr-Tyr-Arg-Pro-Lys-Glu-Asp-Thr-Ala-Asn-Asn- Arg

[0167] V_(L) peptide monomers had no effect on reovirus type 3 receptorexpressions. V_(L)SH peptide specifically down-modulated the expressionof the reovirus type 3 receptor without effecting the expression of Thy1.2 molecules. The effect of V_(L)SH peptide was similar to that of87.92.6 (FIG. 6). The results indicate the specificity of the effect ofV_(L)SH peptide on the reovirus type 3 receptor and confirm thatreceptor aggregation plays a role in the induction of these effects.

[0168] Role of Specific Residues Of V_(L) Peptide Involved In TheInteraction Of V_(L) Peptide With The Reovirus Type 3 Receptor

[0169] Once the shared regions were defined, variant peptides withsubstitutions at several positions in the putative binding domain ofV_(L) peptide were synthesized to study the effect of these forms of thepeptide on cellular physiology. These studies indicate that hydroxylgroups from positions 11 (Tyr), 12 (Ser), 14 (Ser) and 15 (Thr) may beinvolved in directly interacting with the reovirus type 3 receptor. Thisis the region of greatest shared identity of amino acids between theV_(L) peptide and the reo peptide. See Table 1. The variant peptides hadamino acid substitutions at positions 11-16, the region of the V_(L)peptide believed to be the binding domain. To study the effect of theseforms of peptide on cellular physiology, lectin induced mitogenesis wasutilized to provide a system wherein both receptor perturbation (by thepeptides) and aggregation (by the lectin) can be induced.

[0170] Peptide Inhibition of Lymphocyte Proliferation

[0171] Reovirus type 3 and anti-reovirus type 3 receptor antibodies haveboth been demonstrated to inhibit concanavalin A (con A) inducedlymphocyte proliferation (Nepom, J. T. et al., Immunol. Res. 1:255(1982), Sharpe, A. H. and B. N. Fileds, J. Virol. 38:389 (1983),Fontana, A. and H. L. Weiner, J. Immunol. 125:2660 (1980)). The effectsof these peptides on lymphocyte proliferation both in the presence andin the absence of con A have been investigated as follows.

[0172] C3H female mouse spleenocytes were prepared as a single cellsuspension, and cultured with peptides at the concentrations noted inabsence (A) or in the presence (B) of concanavalin A (con A) at 2.5μg/μl. 72 hours later, tritiated thymidine was added, the cells wereharvested 18 hours later and CPM incorporated determined. Per centinhibition was calculated as for FIG. 4. The peptides utilized are thosedescribed for FIG. 4. In the absence of con A, V_(L)SH peptide markedlyinhibited spontaneous lymphocyte proliferation, while V_(L) peptide hadno significant effect (see FIG. 7A) However, in the presence of con A,V_(L)SH peptide and V_(L) peptide had similar effects in inhibitinglymphocyte proliferation (see FIG. 7B).

[0173] As shown in FIG. 8, when variant peptides were utilized lackinghydroxyl groups from positions 12 and 15 (V_(L)A12 and V_(L)A15respectively), the inhibition of con A induced lymphocyte proliferationwas attenuated (FIG. 8A). Lymphocyte proliferation was determined asdescribed above for FIG. 7. The peptides utilized were: V_(L):Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser- Gly-Ser-Thr-Leu-Gln,V_(L)F11: Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser-Gly-Ser-Thr-Leu-Gln, V_(L)A12:Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala- Gly-Ser-Thr-Leu-Gln,V_(L)A13: Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Ala-Ser-Thr-Leu-Gln, and V_(L)A15:Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser- Gly-Ser-Ala-Leu-Gln

[0174] This indicates that these amino acid residues are involved ininteractions critical to receptor perturbation, leading to inhibition ofproliferation. The hydroxyl groups of positions 11 (Tyr) and 14 (Ser)appeared to have less of an effect on this cellular activity (FIG. 8B).

[0175] A peptide with a (Gly-Ala substitution at position 13 in theputative binding domain of V_(L) peptide (V_(L)A13). Also utilized incontrast to the other substitutions described, V_(L)A13 had an increasedeffect on the inhibition of Con A induced lymphocyte proliferation atsome of the concentrations used (FIG. 8B). This V_(L)A13 peptide alsohas increased binding to monoclonal antibody 9BG5, which may mimic thereovirus type 3 receptor on these cells. These studies indicate thatmodification of the V_(L) peptide can identify specific residuesrequired for receptor perturbation, and lead to the development ofvariant peptides with both increased and decreased biologic activity.

[0176] Competitive Binding of 9BG5 to 87.92.6 In The Presence OfPeptides

[0177] Polystyrene wells were coated with purified 87.92.6 or controlIgM, K antibody H022.1 by incubation of purified antibody (purified on agoat anti-mouse IgM column), diluted in 0.1% NaHCO₃ pH 9.5 to 1 μg/mlwith 50 μl/well, overnight at 4° C. The wells were washed, blocked with2% BSA in PBS with 0.1% NaN₃, washed again and a mixture ofradioiodinated 9BG5 and peptides (at the concentrations noted in FIG. 9)were added for one hour at 37° C. The wells were washed and counted. Inall cases, specific CPM bound was determined by subtracting CPM bound toblank wells coated with BSA from CPM bound for 87.92.6 coated wells. Asshown in FIG. 9, binding of ¹²⁵I-9BG5 to wells coated with irrelevantmouse IgM, K antibody H022.1 was similar to binding to blank wells. Percent inhibition was determined by subtracting specific CPM bound in thepresence of inhibitor from specific CPM bound in the absence ofinhibitor, dividing this by CPM bound in the absence of inhibitor, andmultiplying the result by 100. The±SEM of values from two experiments isshown in FIG. 9. V_(L) Peptide Inhibits Binding Of Reovirus Type 3Particles to 9BG5.

[0178] The wells of microtiter plates were coated with neutralizinganti-reovirus type 3 monoclonal antibody 9BG5 or irrelevant classmatched monoclonal All by adsorption to staphylococcal protein A (SPA).SPA (sigma Chemical Co., St. Louis, Mo.) was diluted to 5 μg/ml in 0.1 MNaHCO₃ pH 9.6 and 50 μl/well dispensed into 96 well polystyrene plates.Following overnight incubation at 4° C., the wells were decanted, washedthree times in PBS, and blocked with 2% BSA in PBS with 0.1% NaN₃ forone hour at 37° C. The wells were decanted, washed three times in PBSand monoclonal antibody 9BG5 diluted to 10 μg/ml in 1% BSA in PBS with0.1% NaN₃ was added (50 μl/well) for 1-3 hours at 37° C. Prior studiesindicated that these amounts of SPA and monoclonal murine IgG2aantibodies gave maximal adsorption of antibody on the wells. The wellswere decanted and washed three times in PBS. Competitors were added atthe concentrations noted (100 μl/well) diluted in 0.5% BSA in 5 mMphosphate buffer with 0.45% NaCl and preincubated for 45-60 minutes at23° C. Control experiments indicated that these peptides had no effecton monoclonal antibody binding to the wells. Following preincubationwith inhibitors, radioiodinated reovirus type 3 particles diluted in 1%BSA in PBS with 0.1% NaN₃ were added (5-10×10⁵ CPM per well), and theincubation continued for 45 minutes. Wells were decanted, washed 8-10times with PBS and the CPM bound determined. V_(L) peptide inhibitsbinding of reovirus type 3 particles to 9BG5. As shown in FIG. 10, 6,700CPM were bound to 9BG5 coated wells and 500 CPM were bound to control(All) coated wells in the absence of inhibitors. The mean±standarddeviation of binding inhibition (Determined as noted for FIG. 9) ofreplicate wells is shown. Control peptide B was used in this study. Thecompetitor peptides in FIG. 15 are those described herein. Competitorpeptide V_(L)A6 is identical to V_(L) except that alanine is substitutedfor asparagine at position 6. The competitor peptides inhibited bindingof reovirus type 3 particles to 9BG5.

[0179] V_(L) Peptide Inhibits Binding of Reovirus Type 3 and variant Kto L Cells

[0180] L cells were suspended at 10⁶/ ml in 1% BSA in PBS with 0.1%NaN₃, and 50 μl (5×10⁴) cells) added to each well of a 96-wellmicrotiter plate, and preincubated with inhibitors at the concentrationsnoted for 45-60 minutes at 23° C. Equivalent input CPM of radioiodinatedreovirus type 3, type 1 or variant K particles were added in 50 μl(700,000 to 1,250,000 CPM/well) and incubated for 45 minutes. The cellswere washed three times in 1% BSA in PBS with 0.1% NaN₃ and specific CPMbound determined, as noted in FIG. 9. As shown in FIG. 11 (a) and (b),V_(L) peptide inhibits binding of reovirus type 3 and variant K to Lcells. The mean±S.D. percent inhibition of binding from replicate wellsis shown versus the final concentration of competitor. As shown in FIG.11 (c) and (d), V_(L) variant peptides also inhibit binding of reovirustype 3 to murine L cells.

[0181] Immunization with Peptides Induces Reovirus-Binding Antibodies

[0182] Having established that the V_(L) and reo peptides contain theepitope involved in the interaction between type 3 reovirus and itsspecific cellular receptor, it was decided to test if immunization withthese peptides would induce antibodies capable of interacting withreovirus type 3 and blocking infection. Groups of Balb/c mice wereimmunized with these synthetic peptides as noted in the experimentalprocedures section. Groups of 4 mice received either control peptide inadjuvant, V_(L) peptide coupled to chicken serum albumin (V_(L)-CSA) inadjuvant, V_(H) and V_(L) peptide coupled to CSA (V_(H)+V_(L)-CSA) inadjuvant, reo peptide in adjuvant or reo peptide without adjuvant. As apositive control, an additional group of mice was injected with reovirustype 3. As indicated below, pre-immune serum from these mice disclosedno reovirus neutralizing antibodies indicating no prior exposure tovirus.

[0183] Radioimmunoassay indicated a strong response to the immunizingantigen in all cases (data not shown). Binding of immune serum (day 60)to reovirus type 1 and type 3 is shown in FIG. 12. Specific binding wasdetermined by subtracted CPM bound on a blank plate from CPM bound on avirus coated plate. As a further control, specific binding of normalmouse serum to virus coated plates was also subtracted. To simplifyinterpretation, specific binding is shown for four groups of animals:those immunized with 1) the control peptide, 2) V_(L)-CSA, 3)V_(H)+V_(L)-CSA (all with adjuvant), and 4) reo peptide withoutadjuvant. Mice immunized with reo peptide plus adjuvant made a responsesimilar to those immunized with V_(L)-CSA, V_(H)+V_(L)-CSA plusadjuvant. Mice immunized with type 3 reovirus made a strong response totype 3 virus (specific CPM at a 10⁻³ dilution of serum of 10,428±807)with significant cross-reactivity with type 1 virus (specific CPM at a10⁻³ dilution of 6,976±915). As shown in FIG. 12, serum from miceimmunized with control peptide bound poorly to type 1 or type 3 viruscoated plates at any of the serum dilutions used. In contrast,significant binding of immune serum to type 1 and type 3 virus coatedplates is demonstrated from mice immunized with V_(L)-CSA,V_(H)+V_(L)-CSA or reo peptide. As was expected, binding to type 3 viruswas significantly higher than binding to type 1 virus, although somecross-reactivity is seen. The binding of type 1 virus was likely to havebeen due to some areas of primary sequence similarity between thepeptides used here and the type 1 sigma 1 a protein (Manemitsu, S. M. etal., Biochem. Biophys. Res. Commun., 140:501-510, 1986).

[0184] These results indicate that priming mice with peptides modeledfrom the putative neutralizing epitope of type 3 reovirus or thecorresponding epitope from the anti-receptor monoclonal antibody inducesreovirus binding antibodies.

[0185] Neutralization of Viral Infectivity by Immune Serum from PeptideImmunized Mice

[0186] Serum from peptide immune animals was assayed at three timepoints to evaluate its effects on viral infectivity of L-cells. Twoassays were used to detect neutralization of infectivity. One was adirect cytotoxicity assay measuring the effect of serum on viral lysisof L-cells grown adherent to the wells of 96-well microtiter plates byvital staining, and the other was by measuring inhibition of plaqueformation by serum, with virus and L-cells in soft agar. Results fromthe direct cytotoxicity assay are shown in FIG. 13. Pre-immune serumfrom all of the animals used was assayed and no significant effect ontype 1 or type 3 viral lysis of L-cells was demonstrated. As a positivecontrol, neutralization of L-cell lysis by reovirus was demonstrated byserum from mice immunized with reovirus type 3. This serum producedpotent inhibition of lysis by both type 3 and type 1 virus, although apreferential effect on type 3 viral lysis was noted, with neutralizationtiters of 1:512 for type 3 virus on days 20 and 60, and titers of 1:342and 1:256 for type 1 virus on days 20 and 50 respectively. Serum fromcontrol peptide immunized animals had no effect on L-cell lysis byreovirus type 1 or type 3. Serum from mice immunized with V_(L)-CSA,V_(H)+V_(L)-CSA or reo peptide with or without adjuvant specificallyneutralized L-cell lysis by reovirus type 3 but not type 1 (FIG. 13aversus 13 b). As results were similar for serum from animals immunizedwith reo peptide in the presence or absence of adjuvant, results onlyfrom the latter group is shown. This effect was also seen when serumfrom these mice was assayed for inhibition of plaque formation. In FIG.14, the reciprocal serum titer producing 50% of greater plaqueinhibition is shown for type 1 and type 3 virus from the groupsimmunized with V_(L)-CSA, V_(H)+V_(L)-CSA or reo peptide withoutadjuvant. Again, specific inhibition of plaque formation by type 3 butnot type 1 virus is seen. Since peptide-immune serum specificallyinhibits type 3 but not type 1 viral infectivity, these peptides definethe neutralizing epitope present on reovirus type 3.

[0187] Elicitation of Delayed-Type Hypersensitivity (DTH) to ReovirusType bv Immunization with Peptides

[0188] Prior studies have demonstrated that the specificity of DTHresponses to reovirus infection involved the sigma 1 polypeptide (Weiss,H. L. et al., J. Immunol. 125:278-282, 1980. It was therefore determinedif immunization of mice with these peptides would elicit DTH responsesto intact reovirus. As shown in FIG. 15, significant DTH responses toreovirus type 3 were induced by immunization with V_(L) peptide. Thisresponse was type specific as these animals did not demonstratesignificant DTH responses to reovirus type 1. Use of reassortant virusesmaps the response to the sigma 1 protein. In addition, priming animalswith type 3 virus results in significant DTH to the V_(L) peptide. Atype specific proliferative response to reovirus type 3 in spleen cellsfrom mice immunized with reo peptide was also demonstrated. These dataindicate that V_(L) and reo peptide define an important epitope involvedin T cell-mediated immunity to reovirus type 3.

DISCUSSION

[0189] It has thus been demonstrated that synthetic peptides defined byareas of corresponding sequences between the reovirus type 3 sigma 1polypeptide and a monoclonal anti-receptor antibody 87.92.6 define theepitope on the virus and on the antibody involved in interacting withneutralizing antibody 9BG5, elicit neutralizing antibodies and induceT-cell mediated immunity. In addition it has been shown that one ofthese peptides, V_(L), competes with binding of 87.92.6 to the reovirustype 3 receptor on R1.1 cells. Since 87.92.6 competes with reovirus type3 binding to R1.1 cells (Kauffman, R. S., et al. (1983) supra), it ishypothesized that this epitope in the virus is involved in directlyinteracting with the type 3 reovirus receptor. This is confirmed by theability of V_(L) peptide to inhibit binding of reovirus type 3 to cells.Binding of reovirus type 1 (which utilizes a distinct receptor) is notinhibited, indicating a specific interaction with the reovirus type 3receptor.

[0190] Since this epitope encompasses amino acids 317-332 of the sigma 1polypeptide, this finding would seem at odds with other reports whichhave implicated amino acid 419 of the hemagglutinin in viral resistanceto neutralizing antibodies (Bassel-Duby, R. et al., J. Virol. 60:64-67,1986), and in tissue tropism of the virus (Kaye, K. M. et al., J. Virol.59:90-97, 1986). In those studies, viruses were selected for by growthin the presence of neutralizing antibodies (Spriggs, D. R., and Fields,B. N., Nature (London), 297:68-70, 1982), and those resistant toneutralization by the antibodies had their amino acid sequencedetermined (Bassel-Duby, R., et al, 1986, supra).

[0191] Several possibilities might account for the disparity in theseresults. It is possible that the mutations involving the amino acids 419induce an allosteric effect on the conformation of amino acids 317-332which allows interaction with the viral receptor in the presence of theneutralizing antibodies. In this scenario, amino acids 317-332 would bedirectly involved in binding to the viral receptor and to neutralizingantibody. The mutation at amino acid 419 would induce an allostericalteration in the confirmation of this region that would allow bindingto the viral receptor in the presence of neutralizing antibody. Anotherpossibility is that both regions are involved in binding the viralreceptor. In this case both regions would be in close proximity in thetertiary structure of the sigma 1 polypeptide. This is possible as bothare predicted to be in the “globular head” region of the hemagglutininby computer modeling (Bassel-Duby, R., et al, 1985, supra) . Themutation of 419 would strengthen the interaction of this area of thehemagglutinin with the receptor, thereby overcoming the blockage ofreceptor binding by the neutralizing antibodies binding to residues317-332. While other possibilities exist, clarification of these issuesawaits more detailed knowledge of the tertiary structure of the sigma 1protein.

[0192] These studies have direct implications for vaccine development.It would be greatly desirable to be able to delineate the neutralizingepitopes present on microorganisms to aid in development of syntheticvaccines that would effectively protect individuals from infection,without the risks involved in the use of whole organisms. This would beparticularly useful in situations where there is marked antigenicheterogeneity in the structure of a pathogen, but the binding site forspecific cellular receptors is conserved. A variety of strategies can beand have been employed to determine sites involved in receptor-pathogeninteractions including site-directed mutagenesis and immunization ofanimals with sequential peptides derived from the sequences of pathogenproducts (Elder, J. H., et al, 1987, supra). Site directed mutagenesis,while yielding specific information about sequence variations that leadto differences in biological effects, suffers from the disadvantage thatallosteric effects resulting from the sequence differences could accountfor the effects induced. In this situation, sequence variation in aregion of a gene product may alter the biologic properties of a distantsite and yield misleading information. Analysis of the effects ofantibodies elicited by immunization with sequential peptides derivedfrom pathogen products, while a definitive approach yielding specificinformation, is time-consuming and may require analysis of a largenumber of peptides before a neutralizing immune response is detected.

[0193] The above experiments thus demonstrate a method for producing asynthetic biologically active peptide comprising a sequencecorresponding to a peptide sequence found in corresponding regions ofboth an antigen and in an anti-idiotypic antibody for that antigen. Bydemonstrating corresponding sequences in the sigma 1 cell attachmentprotein of reovirus type 3 and monoclonal anti-receptor antibody 87.92.6the neutralizing epitope of reovirus type 3 was localized. These studiesconfirm that the epitope implicated is the one involved in viral bindingto the cellular reovirus type 3 receptor and in the elicitation ofneutralizing antibodies. Once the shared region has been defined, otherbiologically active peptides can be prepared by modifying this peptidesequence. These modifications are directed to the region believed to beinvolved in binding of the antigen to the receptor. Gly(13) and hydroxylgroups from positions 11(Tyr), 12(Ser), 14(Ser) and 15(Thr) are believedto be involved in directly interacting with the reovirus type 3receptor. Peptide dimers comprising the shared peptide sequence alsohave biological activity and can be shown to have greater affinity thanmonomers.

[0194] As the studies herein indicate, modification of the V_(L) peptidecan lead to development of variant peptides with both increased anddecreased biological activity. Peptide V_(L)A12 has reduced binding toneutralizing monoclonal antibody, reduced binding to the reovirus type 3receptor and reduced biologic activity. Peptide V_(L)A15 has increasedbinding to neutralizing monoclonal antibody, decreased binding to thereovirus type 3 receptor and decreased biological activity. Variantpeptides such as V_(L)A12, if used as immunogens, might prevent aneffective immune response. However, this might be clinically useful insome instances. V_(L) peptide itself, if used as an immunogen, mightelicit an effective immune response, but direct effects of the V_(L)peptide on the retrovirus type 3 receptor might be deleterious to thehost. In this case, a variant peptide such as V_(L)A15, which binds toneutralizing antibodies, but has reduced biologic activity, might beideal as an immunogen as it would elicit neutralizing antibodies butwould not be expected to have significant direct effects on theretrovirus type 3 receptor and would not be expected to be deleteriousto the host. The present approach of defining a shared peptide region ofboth an antigen and an anti-idiotypic antibody (anti-receptor antibody)for that antigen and subsequently modifying this peptide to producepeptides having more or less biological activity is believed to begenerally applicable to other receptor-ligand interactions.

[0195] The present approach further demonstrates a method of immunizinga host mammal against an infectious organism having a site which bindsspecifically to a receptor site on a host cell. This method allowed forthe relatively rapid determination of the neutralizing epitope onreovirus type 3 and is believed to be generally applicable to otherpathogens for which neutralizing immune responses can be demonstrated.

[0196] In the present instance, the reovirus type 3 is known toselectively bind to a structure which is antigenically and structurallysimilar to the mammalian beta-adrenergic receptor. If attachment of apathogen to specific cellular receptors is important in the pathogenesisof infection by that pathogen, the approach outlined here should resultin the ability to determine the oligopeptide epitope involved in thepathogen-receptor interaction. This should also be applicable to otherreceptor-ligand interactions in a more general sense, and in the case ofpolypeptide ligands, should allow the determination of the bindingepitopes involved. It is believed that this strategy will lead to thedevelopment of biologically active compounds that will interact withspecific receptors in predictable ways. Accordingly, a method isdisclosed which is useful for synthesizing biologically active compoundsusing pathogen gene products, such as the reovirus 3, which is known tobind to a physiologic receptor of mammalian cells. Where, as with themammalian reovirus type 3 receptor, the result of such selective bindingis to affect the growth or other metabolic function of the subject cell,the subject method may be used for altering the growth of the mammaliancell by administering the synthetic peptide containing the subjectshared peptide sequence or biologically active modification thereof forthat purpose.

[0197] The strategy of utilizing shared primary structure and molecularmimicry to define interacting oligopeptide epitopes thus should have awide range of applications in the biological sciences to both defineareas of specific interaction between molecules, and to aid in thedevelopment of compounds with predictable biologic activity.

[0198] Those of ordinary skill in this art recognize that variousmodifications can be made in the compounds of the invention withoutdeparting from the scope hereof. For example, peptides of the same class(i.e., conservative substitution as described by Chu et al,“Conformational Parameters For Amino Acids in Helical, Beta Sheet, andRandom Coil Regions Calculated from Proteins”, Biochemistry, 13(2):211,1974, which is incorporated herein by reference) may be substituted inthe sequence shared between the antibody and antigen, provided theactivity of the resulting peptide is not adversely affected. Similarly,it is contemplated that molecular modeling techniques will permitcompounds of quite different primary and secondary structures to besubstituted for the peptides of this invention, provided equivalenttertiary structures, as determined using the methods of this inventionare employed. Additionally, other antibodies, such as otheranti-receptor antibodies to the reovirus type 3 receptor oranti-idiotypic antibodies to neutralizing antibodies may also contactthe receptor using CDR regions. Peptides derived from these regionshaving biologic activity similar to that described herein for V_(L)peptide are also within the scope of the invention.

Example 2

[0199] The present approach also provides an alternative route for thedevelopment and production of biologically active peptides. As shown inFIG. 16, antibodies 15 specific for a receptor 11 of the antigen (orligand) 5 also mimic the antigen 5 in the same way as an anti-idiotypeantibody 9 of the antigen mimics the antigen 5.

[0200] In pathway I, an antibody 7 contains an epitope designatedgenerally 21 complementary to the neutralizing epitope designatedgenerally 19 of antigen 5. This antibody 7 is then used to produce otherantibodies, or anti-idiotype antibodies 9. These anti-idiotypeantibodies 9 will have a region designated generally 23 mimicking theneutralizing epitope 19 of the antigen 5. In pathway II, the receptor 11on cell surface 13 contains an epitope designated generally 25complementary to the neutralizing epitope 19 of the antigen 5; theantibody specific for the receptor 15 will thus contain a regiondesignated generally 27 mimicking the neutralizing epitope 19 of theantigen 5. The anti-receptor antibody 15 is the equivalent ofanti-idiotype antibody 9, since both contain regions (23 and 27)mimicking the neutralizing epitope 19 of the antigen 5. Anti-receptorantibodies 15 can be used as an alternative, or in addition to,anti-idiotype antibodies 9 in the methods described herein to developand produce biologically active peptides 17 with properties of theantigen or ligand.

[0201] Because antigens such as viruses generally contain multipleantigenic epitopes, it may be necessary to screen the antibodiesproduced in response to the inoculation with the ligand, receptor oranti-ligand antibody to select antibodies having specificity for theneutralizing epitope of the antigen. Screening can be done bycompetitive assays that determine the antibody's ability to inhibitbinding of the antigen to the receptor of the cell, those antibodieshaving a greater ability to inhibit binding of the antigen containing ormimicking the neutralizing epitope. Other screening methods includethose as described herein in which a biological function, such asinhibition of DNA synthesis, is triggered. Suitable screening methodsinclude those described herein, and in Burstin, S. J., et al.,Hemagglutinin Virology 117:146-155. It will be obvious to those skilledin the art that various changes to reagents may need to be made in thecompetitive assays when different antigen and receptor pairs are used.

[0202] As demonstrated herein, neutralizing antibody 9BG5, having aspecificity for the antigen HA3 on the reo virus, was used to makeanti-idiotype antibodies having anti-receptor activity. Theseanti-idiotype antibodies also bind to the reovirus type 3 receptor. Theantibodies were screened to identify antibodies that competed orinhibited binding of the neutralizing antibody with the receptor whichwould indicate they contained epitopes that mimic HA3, the antigen. Thevariable region of one antibody having this activity was compared withthe sequence of the antigen HA3 to determine corresponding regions thatdefine the interaction site of HA3 and the receptor.

[0203] Instead of using an anti-receptor antibody that was produced asan anti-idiotype antibody, the receptor itself is also suitable forproducing antibodies that have epitopes mimicking the antigen. Toproduce antibodies by this route, receptor bearing cells are used as animmunogen, as for example in Drebin, et al., Nature (1984) 321:545-547and Drebin, et al., Cell (1985) 41:695-706. Alternatively, purifiedreceptor can be used, as for example in Williams, et al., 1989 J.Neurochem 53:362-369 and Meyers et al., 1992 Receptor 2:1-16, both ofwhich are incorporated herein by reference. These two immunogens can beused to make antibodies, usually monoclonal antibodies, by conventionaltechniques. An animal such as a mouse is first injected with thereceptor, its spleen cells are removed and fused with myeloma cells toform hybridoma cells, the latter are cloned in a serum-containing mediumand the monoclonal antibodies are separated from the medium. Theantibodies are then screened by neutralization assay, as describedabove, to select those antibodies which specifically bind to thereceptor site at the neutralizing epitope. This can be coupled with ascreen that examines the biological effects of receptor binding, forexample the inhibition of DNA synthesis assay described herein. In theexample, both the reovirus and the antibody cause some effect.

[0204] The strategy of utilizing shared primary structure and molecularmimicry to define interacting oligopeptide epitopes thus should have awide range of applications in the biological sciences to both defineareas of specific interaction between molecules, and to aid in thedevelopment of compounds with predictable biologic activity.

[0205] Those of ordinary skill in this art recognize that variousmodifications can be made in the peptides and compounds of the inventionwithout departing from the scope hereof. For example, peptides of thesame class (i.e., conservative substitution as described by Chu et al,Biochemistry, 13(2):211, 1974, which is incorporated herein byreference) may be substituted in the sequence shared between theantibody and antigen, provided the activity of the resulting peptide isnot adversely affected. Similarly, it is contemplated that molecularmodeling techniques will permit compounds of quite different primary andsecondary structures to be substituted for the peptides of thisinvention, provided equivalent tertiary structures, as determined usingthe methods of this invention are employed. Additionally, otherantibodies, such as other anti-receptor antibodies to the reovirus type3 receptor or anti-idiotypic antibodies to neutralizing antibodies mayalso contact the receptor using CDR regions. Peptides derived from theseregions having biologic activity similar to that described herein forV_(L)peptide are also within the scope of the invention.

Example 3

[0206] Design of Immunogenic Human Immunodeficiency Virus Peptide

[0207] HIV, the AIDS virus, enters its target cell in a series of steps.The first event in this sequence is the attachment of the viral envelopegp120 protein to CD4 on the surface of the target cell. In oneembodiment of the invention, gp120 is the protein of interest, that is,the pathogen antigen. Computer graphics and comparative molecularmodelling may be used to study the potential conformational propertiesof the CD4 binding site on gp120.

[0208] In the comparative modelling approach, the structure of anunknown protein is deduced from sequence similarities between portionsof crystallographically known proteins and the protein fragment to bemodelled (Greer et al., 1989, Prog. Clin. Biol. Res. 289:385-397). Thisapproach has been used in the modelling of a wide range of proteinsincluding antibodies and T-cell receptors (De la Paz et al., 1986, EMBOJ. 5:415-425; Chothia et al., 1988, EMBO J. 7:3745-3755). It has beenhypothesized that retroviruses are evolving towards structurallymimicking epitopes of the immunoglobulin (Ig) superfamily (Oldstone,M.B.A., 1987, Cell 50:819-820) that interact with normal immunestructures like CD4.

[0209] Structural examination of antigen combining regions of T cellreceptors and antibodies indicates that the recognition sites of thisparticular superfamily class are organized reverse turns or loops. ForMHC molecules, molecular recognition areas are highly α helical Bjorkmanet al., 1987, Nature 329:506-512). Structural analysis of ananti-receptor antibody that mimics the cell attachment site of reovirushemagglutinin affirms the possibility of shared β-type conformation asone underlying recognition feature bestowing mimicking properties onantibodies (Williams et al., 1988, Proc. Natl. Acad. Sci. USA,85:6488-6492). In this context, an anti-CD4 antibody that competes withgp120 for the same CD4 binding site is considered an anti-receptor(anti-idiotypic) antibody that mimics gp120 (McDougal et al., 1986,Immunol. 137:2937-2944).

[0210] In the deduction of the possible topography for the CD4 bindingsite, one may first-examine the Protein Sequence Database (Devereux etal., 1984, Nucl. Acids Res. 12:387-395) using overlapping sequences ofthe BH10 isolate of HIV from residue 343 through 511. Optimal sequencealignment of the putative cell attachment site of gp120 with members ofthe Ig superfamily imply a degree of similarity between the site andantibody complementarity determining regions (CDRs) (FIG. 18).

[0211] The degree of similarity between the 383-455 region and CDR loopsimplies only that contact regions between gp120 and CD4 may exhibit βturns or loops, and not that gp120 itself folds like an immunoglobulin.This is evident because the intervening residues between the perceived βloops in gp120 are very different from those in antibodies. Thedisulfide bridge connecting residues 418 and 445 may preserve theanti-parallel β strands with a reverse turn geometry. In antibodystructures, CDR1 and CDR3 of light chains pack against each other andare stabilized by a disulfide bond. If this type of structure exists ingp120, then the β loop comprising residues 419-429 would be packed withthe β loop comprising residues 446-454; this packing may be stabilizedby the disulfide bridge formed between amino acids 418 and 445. In thismodel, residues 430-438, representative of a β structure, would still beexposed for contacting CD4.

[0212] A molecular model for the region 413-455 (FIG. 19) may beconstructed based upon these structural concepts, utilizing alight-chain antibody structure known as REI (Bernstein et al., 1977,Mol. Biol. 112:535-542) as a template for the first and thirdhypervariable region, in which the cysteines in gp120 at positions 418and 445 form a disulfide bridge. The cysteines are positionallyconserved with respect to each other as in the light chain. The modelmay be energy optimized using molecular mechanics and dynamics. Themodel depicted in FIG. 18 indicates that the residues 421-438 define acentral turn region of the domain that may be surface exposed forinteraction with CD4. Neutralizing antibodies have been shown to bedirected toward this site (Sun et al., 1989, J. Virol. 63:3579-3585).The in vitro biological activity of the 421-438 peptide fragment inmodulating CD4-depending cellular function and this fragment's abilityto induce an anti-HIV response is as described below. This peptide wasshown to block virus binding and appears to exhibit cellular regulatoryfunctions such as immunosuppression that parallels gp120.

[0213] The putative CD4 binding domain of the human molecule (Lasky etal., 1987, Cell 50:975-985) was modelled after an immunoglobulinvariable region. A topography of the purported gp120 binding site forCD4 was obtained based upon a comparative modelling approach utilizingcorresponding sequences with the immunoglobulin superfamily(Kieber-Emmons et al., 1989, Biochem. Biophys. Acta 989:281-300).Molecular modeling of the 415-456 region of gp120 suggested that adisulfide bond can be formed by the cysteine residues at positions 418and 445. The model indicates that the residues 421-438 define a centralturn of the domain that is surface-exposed for possible interaction withCD4. Monoclonal antibodies that map to the 421-437 region have recentlybeen shown to have anti-HIV neutralizing ability (Sun et al., 1989, J.Virol. 63: 3579-3585). The 421-438 linear peptide termed B138 (Tables IIand III) was synthesized, purified, and assayed for its immunoreactivitywith human sera and its ability to induce an anti-HIV response.

[0214] The reactivity of linear B138 with sera (1:4 dilution) fromhealthy HIV-seronegative and seropositive study subjects has beenassessed. Low levels of reactivity to B138 were detected using sera fromnineteen HIV-seropositive, asymptomatic individuals, with only 2 of 18(11%) generating levels of anti-B138 antibodies that were significantlydifferent (p<0.003) from HIV-seronegative controls. Thirty-three percent(7 of 21) of ARC patients and 10% (2 of 20) of AIDS patients hadsignificant levels of antibodies that bound B138. Only one HIV-infectedindividual, an ARC patient, had levels of anti-B138 antibodies that weresignificantly different from HIV-seronegative individuals at 1:32dilutions of serum (p<0.003). These results demonstrate that HIV-1 doesnot stimulate the production of antibodies that bind B138 in mostinfected individuals. Furthermore, in the few individuals that haddetectable levels of anti-B138 antibodies, these antibodies occurred atrelatively low titers and did not correlate with neutralizing orprotective activities.

[0215] The immunogenicity of B138 was determined by subcutaneousimmunization of mice and rabbits. Mouse antisera to B138 was shown topreferentially bind to HIV_(HTLVIIB)-infected H9 cells as assessed byflow cytometry analysis. Rabbit anti-B138 sera could immunoprecipitatepurified recombinant glycosylated gp120 as assessed by radiolabelledimmunoprecipitations, albeit weakly.

[0216] However, the immunogenicity of B138 increased dramatically whenthe B138 was made into the cyclic peptide, 1005-45 (Tables II and III)by cysteine bond formation. Molecular dynamic calculations of the linear421-438 segment and the cyclic 418-445 fragment suggested that theconformational populations available to the 421-438 region under thecyclic constraints were restricted and more similar to our predictedgeometry of the gp120 putative CD4 binding epitope. Radiolabelledimmunoprecipitations (RIPs) of rabbit anti-sera against the cyclicpeptide showed significant specificity towards recombinant glycosylatedgp120 (FIG. 20). The reactivity is similar to that of theimmunoreactivity of polyclonal human HIV positive sera reactive withgp120. Infectivity assays with rabbit anti-B138 and rabbit anti-1005/45sera showed that both peptides could elicit neutralizing antibodies.

[0217]FIG. 21 shows the results of experiments in which rabbits wereimmunized with various gp120-derived peptides (see Table II) conjugatedto Keyhole limpet hemocyanin (KLH) by glutaraldehyde fixation. Antiserawas obtained and tested for binding to recombinant HIV-1 gp120peptide-coated radioimmunoassay plates (using 1 μg/ml gp120 at 50μl/well, in 0.1 M NaHCO₃ and incubating overnight at 4° C). Theindicator antibody was ¹²⁵I-labelled anti-rabbit immunoglobulinantibody. FIG. 21 indicates that the highest levels of bound antibodywere associated with antisera produced by animals immunized with 1005-45cyclic peptide as compared to lower levels of bound antibody associatedwith antisera produced by animals immunized with linear B138. Higherbound levels of antibody could result from a generally greater immuneresponse and/or the production of antibodies that have a higher affinityfor gp120.

[0218] The invention is not limited in scope by the embodimentsdisclosed in the examples which are intended as illustrations of a fewaspects of the invention and any embodiments which are functionallyequivalent are within the scope of this invention. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art and are intendedto fall within the scope of the appended claims.

Example 4

[0219] As an alternative example, CD4 may be the protein of interest.

[0220] Interestingly, the CD4 receptor is only one of a small number ofviral receptors that have been cloned (White et al., 1989, Cell56:725-728). An examination of the DNA sequence and the exon/intronorganization of the nucleotides which encode the CD4 glycoproteinstrongly suggests that this gene has evolved from a primordial Ig gene(see Littman, D. R., 1987, Annu. Rev. Immunol. 5:561-584 for review).Similarity profiles of the CD4 amino-acid sequence also reflect thisrelatedness to members of the Ig superfamily (FIG. 22; for reviews ofthe superfamily see Hood et al., 1985, Cell 40:225- 229 and Williams etal., 1988, Annu. Rev. Immunol. 6:381-405). The most striking similaritybetween the CD4 protein and the Ig superfamily occurs between the first84 amino acids of the CD4 glycoprotein and the Ig light chain variabledomain (FIG. 22). Although other CD4 regions also show similarity,secondary structure predictions generated from this domain of CD4 arealso extraordinarily similar to those observed for the Ig light chainvariable domains. Membership of the CD4 protein in the superfamilyimplies that the structure of the Ig-like domains of this protein shouldexhibit the classical “Ig-fold” (Schiffer et al., 1973, Biochemistry12:4620-4631; Poljak et al., 1973, Proc. Natl. Acad. Sci. U.S.A.70:3305-3310; Amzel et al., 1979, Annu. Rev. Biochem. 48:961-997).Therefore the alignment of CD4 with members of the Ig superfamilyindicates tertiary structural similarities when residues, identified byX-ray diffraction studies of antibody hypervariable regions, are alignedon the premise that the basic building-block structure and interactionhave been conserved (FIG. 23).

[0221] The sequence relationship between CD4 and immunoglobulins allowsfor general conceptions about the structure of CD4 to be formulated andcorrelated with CD4 epitope mapping studies. Analysis of Ig structure todelineate possible unique epitopes may therefore be used to examinegp120 binding to CD4. Structural analysis of epitope locations on thesurfaces of antibodies suggests that there are separate ornon-overlapping (epitope) recognition sites that involve both classicalCDR and framework regions (Kieber-Emmons et al., 1986, Immunol. Rev.90:29-48; FIG. 23). Such regions have been referred to as idiotypedetermining regions (IDR) (Kieber-Emmons et al., 1986, Immunol. Rev.90:29-48). Each of these epitope (putative recognition) sites may haveunique functional properties. By inference, the CD4/Ig superfamilysequence alignment implies that HIV, MHC class II, ancillary proteinssuch as CD3, and T cell receptors may bind to CD4 in noncompetitiveways.

Example 5

[0222] Design of a Cyclic Peptide which binds to the Cellular ReovirusReceptor and can block the interaction between Reovirus and its TargetCells

[0223] In this embodiment of the invention, the protein of interest isan immunoglobulin molecule, and the method of the invention comprisesidentifying a region of the molecule which is similar to the CDR ofanother immunoglobulin, synthesizing peptides which comprise portions ofthe identified CDR, and then modifying the peptide such that it hasbiological activity. This example presents a nonlimiting working exampleof an embodiment in which a cyclic peptide is designed to resemble a CDRof an anti-virus receptor antibody.

MATERIALS AND METHODS

[0224] Peptides

[0225] All peptides were synthesized by solid phase methods, deprotectedand released from the resin utilizing anhydrous HF. Peptides werelyophilized and further purified by high performance liquidchromatography utilizing a TSK 3000 column and lyophilized. Purity wasassessed by high performance liquid chromatography utilizing a C-6column and a 0-70% acetonitrile gradient. All peptides were greater than90% pure. Peptides (containing internal cysteine residues) were cyclizedfor experiments by dissolving them at 2 mg/ml in distilled water, andstirring them overnight exposed to the air. The peptides had no freesulfhydrlys following this procedure by Ellman determination.

[0226] Reovirus

[0227] Purified reovirus type 3 was prepared and radioiodinated usingmethods set forth in Williams et al., 1988, Proc. Natl. Acad. Sci. USA85:6488-6492, which is incorporated herein by reference.

[0228] Monoclonal Antibodies

[0229] Monoclonal antibodies 9BG5, which binds to reovirus type 3hemagglutinin, and 87.92.6, which mimics the reovirus type 3hemagglutinin by binding to both 9BG5 as well as the reovirus type 3receptor, are as described in Williams et al., 1989, Proc. Natl. Acad.Sci. U.S.A. 86:5537-5541, which is incorporated herein by reference.

Determination of Free Sulfhydryls in Peptides

[0230] (Ellman Determination)

[0231] Peptides dissolved in dH₂O at 2 mg/ml were added at 5, 10, or 20μl to 10 mM NaPO₄ pH 7.0 for a final volume of 1 ml. To this was added 6μl of 2,2′-bis azidothiobenzoic acid (ATBS, Sigma Chemical Co., St.Louis, Mo.) in 50 mM NaPO₄ pH 8.0. This was allowed to react for greaterthan 3 minutes and the optical density (OD) at 420 nm was subsequentlydetermined.

[0232] Radioimmunoassay (RIA)

[0233] RIA plates (Dynatech Laboratories, Alexandria, Va.) were coatedwith peptides by evaporation of varying amounts of peptides in distilledwater overnight at 37° C. The wells were washed with PBS, blocked with2% bovine serum albumin (BSA) in PBS with 0.1% NaN₃, and washed withPBS. Partially purified 9BG5 [(NH4)₂SO₄ precipitate] was added atvarying dilutions for greater than 1 hour at 37° C. The wells werewashed in PBS and 50,000-100,000 counts per minute of ₁₂₅I-labelled goatanti-mouse light chain (anti-k & anti-[Sigma] iodinated by chloramine T)was added per well in 1% BSA in PBS for 1-2 hours at 37° C. The wellswere decanted, washed extensively, and CPM bound determined. SpecificCPM bound was determined by subtracting the CPM bound to uncoated wellsfrom the CPM bound to peptide coated wells.

[0234] Competitive RIA

[0235] RIA plates were coated with Staphylococcus protein A (SigmaChemical Co., St. Louis, Mo.) by incubation of 50 μl per well of a 5μg/ml solution overnight at 4° C. The wells were washed with PBS,blocked with 2% BSA/PBS/0.1%NaN₃, and purified 9BG5 or isotype matchedcontrol monoclonal (All) adsorbed to the wells by incubation of 50 μl ofa 10 μg/ml solution (purified antibody) in 1% BSA/PBS/NaN₃ for 1-2 hoursat 37° C. The wells were washed, and competitors were added at variousconcentrations in 100 μl of 0.5% BSA/0.45% NaCl/0/05% phosphate bufferpH 7.2 for 1 hour at 37° C. ¹²⁵I-labelled reovirus type 3 (5−10×10⁵ CPMper well) or unlabelled antibody (87.92.6 or isotype matched monoclonalE4.49.2) at a 1:100 dilution of ascites in 1% BSA/PBS/0.1% NaN₃) wasadded for an additional 30-45 minutes at 23° C. For wells incubated with87.92.6, the wells were washed in PBS, and ¹²⁵I-labelled goat anti-mouseIg added for an additional 60 minutes at 37° C. The wells were washedextensively, and CPM bound determined. For reovirus binding, specificCPM bound was determined by subtracting CPM bound to All coated wellsfrom CPM bound to 9BG5 coated wells. For 87.92.6 binding, specificbinding was determined by subtracting CPM bound following incubationwith E4.49.2 ascites from CPM bound following 87.92.6 incubation. %inhibition binding was determined by the formulae:

[(Specific CPM bound w/o Inhibitor)−(Specific CPM bound withinhibitor)×100]/Specific CPM bound without inhibitor.

[0236] That is, the amount of specific CPM bound w/o Inhibitor minus theamount of specific CPM bound with inhibitor, the total amount remainingbeing multiplied by 100, the product of which is divided by specific CPMbound without inhibitor.

[0237] Inhibition of Viral Binding to Cells

[0238] The cells were centrifuged and washed twice in 1% BSA/PBS/0.1%NaN₃. 5×10⁴ cells or 1.25×10⁶ R1.1 cells in 50 μl were distributed in 2%BSA/PBS/NaN₃ blocked RIA wells. For peptide studies, 50 μl of inhibitorwas added in dH₂O to the cells. Following a 30 minute incubation, Lcells and ¹²⁵I-labelled reovirus type 3 were combined for an additional30 minutes at 37° C. The cells were spun, washed three times in ice coldPBS, and specific CPM bound was determined as noted above. Percentinhibition of binding was calculated by the formulae above.

[0239] Flow Cytometry Analysis

[0240] The ability of peptides to inhibit antibody binding to cells wasdetermined by preincubation of the cells with varying amounts ofinhibitor (in 100 μl dH₂O) for between 30 minutes and 1 hour at 23° C.Cells (either L cells or R1.1 cells) were washed in 1% BSA/PBS/0.1%NaN₃, and resuspended at 10⁷/ml. 100 μl of cells were then added in 1%BSA/PBS/0.1% NaN₃, and the incubation continued for 20-30 minutes.Antibodies (5 or 10 μl were added for an additional 20 minutes at 23° C.Ice cold 1% BSA/PBS/0.1% NaN₃, was added, the cells centrifuged andwashed prior to counterstaining with a 1:100 dilution of FITC goatanti-mouse Ig (Fisher Scientific) in 1% BSA/PBS/0.1% NaN₃. The cellswere washed twice and fluorescence intensity determined. Inhibition ofbinding was calculated as noted above with mean channel number utilizedin place of CPM.

[0241] Coupling of Peptides to KLH and Immunization

[0242] Peptides were coupled by glutaraldehyde fixation or specificcoupling through a heterobifunctional cross-linker (MBS, Pierce ChemicalCo.) (Romano et al., 1989, J. Neurochem. 53:362-369). Immunization wasas described in Romano et al., supra.

RESULTS AND DISCUSSION

[0243] Peptide Cyclization

[0244] One measure of the optimal folding conformation of V_(L) peptideis reflected by the ability of cysteine-containing variates to cyclize.If the cysteine residues are placed in various positions in one or theother side of a predicted reverse turn, the residues placed in the mostenergetically favorable locations for assuming a reverse turn structureshould also cyclize most rapidly. This can be established utilizingseveral cysteine containing peptides as outlined in Table III.

[0245] These peptides were subjected to oxidation by agitating asolution (2 mg.ml in 0.1 M NaHCO₃) at 37° C. for varying periods of timeexposed to air. The disappearance of free sulfhydryls was quantitated byEllman determination as above, and % loss of sulfhydryls with timecalculated. The results are shown in FIG. 23.

[0246] Peptides (Table III) were agitated at 37° C. for varying periodsof time and loss of sulfhydryls quantitated. As noted, V_(L)C₆C₁₆ andV_(L)C₉C₁₆ had the most rapid loss of sulfhydryls in this assay, whileV_(L)C₁₀C₁₆ peptide forms intramolecular disulfide bridges more slowlythan the other two peptides, and implies that the corresponding cyclicconformation of V_(L)C₁₀C₁₆ may be energetically more costly to assumethan that of V_(L)C₈C₁₆ or V_(L)C₉C₁₆.

[0247] The oxidation of these peptides did not necessarily imply thatcyclic peptide formation was taking place, as intermolecular disulfidebridges also might have been forming. This issue was also examined byexamining reduced (2-mercaptoethanol treated) and non-reduced variatesof these peptides by size-exclusion chromatography utilizing a SephadexG-10 superfine column. These studies indicated that both V_(L)C₈C₁₆ andV_(L)C₉C₁₆ peptides remained chiefly as monomers following oxidation,while a sizeable proportion of V_(L)C₁₀C₁₆ migrated more rapidlyfollowing oxidation. This indicates that the V_(L)C₁₀C₁₆ is formingintermolecular disulfide bridges, with subsequent formation of highermolecular weight forms. In contrast, V_(L)C₈C₁₆ and V_(L)C₉C₁₆ did notform intermolecular disulfide bridges, indicating that these peptidesmore readily fold into an appropriate conformation for intramoleculardisulfide bond formation.

[0248] Binding of 9B.G5 to Peptides

[0249] To assess the optimal conformation for binding of the V_(L)peptide analogs, they were utilized to coat radioimmunoassay (RIA)plates, and 9B.G5 bound by standard RIA procedures. The results areshown in FIG. 24.

[0250] As can be seen, binding to V_(L)C₉C₁₆ peptide was higher thanbinding to the other cyclic V_(L) peptide analogs. This indicates thatV_(L)C₉C₁₆ peptide has enhanced binding to 9B.G5 on solid phase RIA incomparison with the other cyclic peptides. Inhibition of 9B.G5-87.92.6Interaction by Peptides.

[0251] While the solid phase RIA indicates a higher affinity of 9B.GS toV_(L)C₉C₁₆ peptide compared to the other cyclic peptides, it does notaddress the affinity of this interaction in solution. To investigate theoptimal solution conformation for 9B.G5 binding, the peptides wereutilized to inhibit 9B.G5-87.92.6 interaction in a liquid phase assay.As shown in FIG. 25, the results of this assay indicate the V_(L)C₉C₁₆peptide again demonstrates a higher affinity of interaction comparedwith the other cyclic peptide variants. When compared with a linearanalog of V_(L) peptide (FIG. 26), V_(L)C₉C₁₆ peptide displays anincreased affinity of binding (40 fold higher affinity). This indicatesthat the increased conformational stability of this cyclic peptideincreases its binding affinity for 9B.G4. It is also demonstrated that apeptide derived from the 81.92.6 heavy chain variable region CDR II(V_(H) peptide) is able to inhibit the 87.92.6-9B.G5 interaction. Whilethis peptide inhibits the idiotype- anti-idiotype interaction, it doesnot significantly interact with the reovirus type 3 receptor (Reo3R).

[0252] Inhibition of 9B.GS-Reovirus type 3 Interaction by Peptides

[0253] To confirm that V_(L)C₉C₁₆ peptide represents an optimalconformation for 9B.G5 binding in solution phase it was utilized toinhibit binding of ¹²⁵I-labelled reovirus type 3 to 9B.G5 in a similarassay. The results are shown in FIG. 27. As can be seen, V_(L)C₉C₁₆peptide also exhibited higher affinity than the other cyclic peptides inthis assay. When compared with linear V_(L) peptide and dimeric V_(L)SHpeptide (FIG. 28), V_(L)C₉C₁₆ peptide demonstrates higher affinity thanlinear V_(L) peptide, and similar affinity on a molar basis as dimericV_(L)SH peptide.

[0254] Inhibition of REO3R-87.92.6 Interaction by Peptides

[0255] To assess the affinity of the cyclic peptides for the reovirustype 3 receptor (Reo3R), they were utilized in a series of assays toinhibit binding of 87.92.6 or control antibodies to specific receptors.As shown in FIG. 29, V_(L)C₉C₁₆ peptide inhibited binding of 87.92.6 tomurine L cells and R1.1 thymoma cells. In contrast, linear V_(H) peptidehad no effect on 87.92.6 binding, while V_(L) peptide is a lesseffective competitor on L cells, and ineffectual on R1.1 cells. Theinhibition by V_(L)C₉C₁₆ peptide was specific as binding of isotypematched monoclonal H013.4 to Thy1.2 molecules was not inhibited byV_(L)C₉C₁₆ peptide (FIG. 30). Thus, V_(L)C₉C₁₆ peptide is a specificReo3R ligand with enhanced affinity compared with its linear analog.

[0256] Inhibition of REO3R-Reovirus type 3 Interaction by Peptides

[0257] To further evaluate the interaction of V_(L)C₉C₁₆ peptide withthe Reo3R, the peptide was utilized to compete with ¹²⁵I-labelledreovirus type 3 for binding to the Reo3R. As indicated in FIG. 31,V_(L)C₉C₁₆ peptide demonstrated higher affinity for the Reo3R thanV_(L)C₈C₁₆ peptide or V_(L)C₁₀C₁₆ peptide. When compared with linearV_(L) peptide (FIG. 30), V_(L)C₉C₁₆ peptide demonstrated 40 fold higheraffinity for the Reo3R, and similar affinity to dimeric V_(L)SH peptide.

[0258] This confirms that cyclic analogs of V_(L) peptide demonstratehigher affinity of binding to the Reo3R than the linear peptide analogs.This strategy should be applicable to peptides derived from otherantibody variable regions, and defines an overall strategy fordetermining the optimal conformation for binding of these peptides.TABLE I Synthetic Peptides Comprising Corresponding Sequences of 87.92.6and the Reovirus Type 3 Hemagglutinin V_(H) Gln Gly Leu Glu Trp Ile GlyArg Ile Asp Pro Ala Asn Gly Reo Gln Ser Met --- Trp Ile Gly Ile Val SerTyr Ser Gly Ser Gly Leu Asn V_(L) Lys Pro Gly Lys Thr Asn Lys Leu LeuIle Tyr Ser Gly Ser Thr Leu Gln Control Lys Ser Gly Asn Ala Ser Thr ProGln Gln Leu Gln Asn Leu Thr Leu Asp Ile Arg Gln Arg

[0259] TABLE II Peptide Number Sequence 466 FRPGGGDMRDNWSEL 1005-45CRIKQFINMWQEVGKAMYAPPISGQIRC B138 KQFINMWQEVGKAMYAPP

[0260] TABLE III Peptides Utilized in These Studies Designation SequenceV_(L): Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr LeuGln V_(L)SH: Cys Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly SerThr Leu Gln V_(L)C₈C₁₆: Lys Pro Gly Lys Thr Asn Lys Cys Leu Ile Tyr SerGly Ser Thr Cys Gln V_(L)C₉C₁₆: Lys Pro Gly Lys Thr Asn Lys Leu Cys IleTyr Ser Gly Ser Thr Cys Gln V_(L)C₁₀C₁₆: Lys Pro Gly Lys Thr Asn Lys LeuLeu Cys Tyr Ser Gly Ser Thr Cys Gln B138: Lys Gln Phe Ile Asn Met TrpGln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro 1005/45: Cys Arg Ile Lys GlnPhe Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile SerGly Gln Ile Arg Cys

1. A method of constructing a compound capable of eliciting in a mammal,a neutralizing immune response against a pathogen comprising the stepsof: a) generating a neutralizing antibody specific for an epitope of anantigen of said pathogen; b) generating an anti-idiotypic antibodyspecific for said neutralizing antibody; c) comparing amino acidsequences of said anti-idiotypic antibody and said epitope; and d)determining an amino acid sequence that corresponds to an amino acidsequence of said anti-idiotypic antibody and also corresponds to anamino acid sequence of said epitope.
 2. The method of claim 1 furthercomprising: e) synthesizing a peptide comprising an amino acid sequencethat corresponds to an amino acid sequence of said anti-idiotypicantibody and also corresponds to an amino acid sequence of said epitope.3. The method of claim 1 wherein said pathogen is a reovirus and saidantigen is a haemagluttin sigma
 1. 4. The method of claim 1 wherein saidpathogen is HIV and said antigen is a gp120.
 5. The method of claim 1wherein said amino acid sequence of said anti-idiotypic antibody is anamino acid sequence of a complementarity determining region.
 6. Themethod of claim 2 further comprising the step of: modifying saidsynthesized peptide to alter the three dimensional conformation thereof;and/or modifying synthetic peptides by attaching connectors thereto andmaintaining said synthetic peptides under conditions selected to allowformation of peptide dimers.
 7. A method of constructing a compoundcapable of preventing a biologically active protein or a pathogen frombinding to a receptor which comprises the steps of: a) generating ananti-receptor antibody capable of preventing said biologically activeprotein or said pathogen from binding to said receptor; b) comparingamino acid sequences of said anti-receptor antibody and saidbiologically active protein or an antigen of said pathogen; and c)determining an amino acid sequence that corresponds to an amino acidsequence of said anti-receptor antibody and also corresponds to an aminoacid sequence of said biologically active protein or said antigen ofsaid pathogen.
 8. The method of claim 7 further comprising: d)synthesizing a peptide comprising an amino acid sequence thatcorresponds to an amino acid sequence of said anti-receptor antibody andalso corresponds to an amino acid sequence of said biologically activeprotein or said antigen of said pathogen.
 9. The method of claim 7wherein said pathogen is a reovirus and said receptor is abeta-adrenergic receptor-like structure which binds to haemagluttin andsaid antigen is a reovirus haemagluttin sigma
 1. 10. The method of claim7 wherein said pathogen is HIV, said receptor is a CD4 molecule and saidantigen is a gp120.
 11. The method of claim 7 wherein said amino acidsequence of said anti-receptor antibody is an amino acid sequence of acomplementarity determining region.
 12. The method of claim 8 furthercomprising the step of: modifying said synthesized peptide to alter thethree dimensional conformation thereof; and/or modifying syntheticpeptides by attaching connectors thereto, and maintaining said syntheticpeptides under conditions selected to allow formation of peptide dimers.13. A method of constructing a compound capable of preventing a pathogenor a biologically active protein from binding to a receptor whichcomprises the steps of: a) generating an antibody specific for saidbiologically active protein or an antigen of said pathogen, saidantibody being capable of preventing said biologically active protein orsaid pathogen from binding to said receptor; b) generating ananti-idiotypic antibody specific for said antibody; c) comparing aminoacid sequences of said anti-idiotypic antibody and said biologicallyactive protein or said antigen; and d) determining an amino acidsequence that corresponds to an amino acid sequence of saidanti-idiotypic antibody and also corresponds to an amino acid sequenceof said biologically active protein or said antigen.
 14. The method ofclaim 13 further comprising: e) synthesizing a peptide comprising saidamino acid sequence that corresponds to an amino acid sequence of saidanti-idiotypic antibody and also corresponds to an amino acid sequenceof said biologically active protein or said antigen.
 15. The method ofclaim 13 wherein said pathogen is a reovirus and said receptor is abeta-adrenergic receptor-like structure which binds to haemagluttin andsaid antigen is a haemagluttin sigma
 1. 16. The method of claim 13wherein said pathogen is HIV, said receptor is a CD4 molecule and saidantigen is a gp120.
 17. The method of claim 13 wherein said amino acidsequence of said anti-idiotypic antibody is an amino acid sequence of acomplementarity determining region.
 18. The method of claim 14 furthercomprising the step of: modifying said synthesized peptide and alteringits three dimensional conformation; and/or, modifying synthetic peptidesby attaching connectors thereto, and maintaining said synthetic peptidesunder conditions selected to allow formation of peptide dimers.
 19. Amethod of constructing a biologically active compound comprising thesteps of: a) generating an anti-receptor antibody capable of effectingan activity or function of a cell; b) comparing amino acid sequences ofsaid anti-receptor antibody and an antigen which binds to said receptoror a biologically active protein which binds to said receptor; and c)determining an amino acid sequence that corresponds to an amino acidsequence of said anti-receptor antibody and also corresponds to an aminoacid sequence of said antigen or an amino acid sequence of saidbiologically active protein.
 20. The method of claim 19 furthercomprising: e) synthesizing a peptide comprising an amino acid sequencethat corresponds to an amino acid sequence of said anti-receptorantibody and also corresponds to an amino acid sequence of said antigenor an amino acid sequence of said biologically active protein.
 21. Themethod of claim 19 wherein said antigen is from a reovirus and saidreceptor is a beta-adrenergic receptor-like structure which binds tohaemagluttin and said antigen is a haemagluttin sigma
 1. 22. The methodof claim 19 wherein said amino acid sequence of said anti-receptorantibody is an amino acid sequence of a complementarity determiningregion.
 23. The method of claim 20 further comprising the step of:modifying said synthesized peptide and altering its three dimensionalconformation; and or, modifying synthetic peptides by attachingconnectors thereto and, maintaining said synthetic peptides underconditions selected to allow formation of peptide dimers.
 24. Asynthetic biologically active peptide consisting essentially of an aminoacid sequence corresponding to an amino acid sequence of ananti-idiotypic antibody or an anti-receptor antibody and alsocorresponding to an amino acid sequence of an antigen or biologicallyactive protein.
 25. The peptide of claim 24 wherein said antigen is froma haemagluttin sigma 1 from a reovirus.
 26. The peptide of claim 24wherein said antigen is gp120 from HIV.
 27. The peptide of claim 24wherein said amino acid sequence of an anti-idiotypic antibody oranti-receptor antibody is an amino acid sequence of a complementaritydetermining region.
 28. The peptide of claim 24 comprising an amino acidsequence selected from the group consisting of:Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser- Gly-Ser-Thr-Leu-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala- Gly-Ser-Thr-Leu-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-LeU-Leu-Ile-Tyr-Ser- Ala-Ser-Thr-Leu-Gln;and Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Ala-Leu-Gln.


29. The peptide of claim 24 wherein said peptide has an amino acidsequence consisting essentially of:Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser- Gly-Ser-Thr-Leu-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala- Gly-Ser-Thr-Leu-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser- Ala-Ser-Thr-Leu-Gln; orLys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser- Gly-Ser-Ala-Leu-Gln.


30. The peptide of claim 24 comprising an amino acid sequence selectedfrom the group consisting of:Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser- Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Cys-Leu-Ile-Tyr-Ser- Gly-Ser-Thr-Cys-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Cys-Ile-Tyr-Ser- Gly-Ser-Thr-Cys-Gln;and Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Cys-Tyr-Ser-Gly-Ser-Thr-Cys-Gln.


31. The peptide of claim 24 having an amino acid sequence consistingessentially of: Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln; Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Cys-Leu-Ile-Tyr-Ser- Gly-Ser-Thr-Cys-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Cys-Ile-Tyr-Ser- Gly-Ser-Thr-Cys-Gln; orLys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Cys-Tyr-Ser- Gly-Ser-Thr-Cys-Gln.


32. The peptide of claim 24 wherein said peptide is a dimer comprising afirst peptide sequence and a second peptide sequence, wherein: saidfirst peptide sequence and said second peptide sequence may be the sameor different; each of said first peptide sequence and said secondpeptide sequence are identical or similar to similar portions of anantigen and an anti-idiotypic antibody for said antigen; and, said dimeris joined at one end.
 33. The peptide of claim 24 wherein said peptideis a dimer comprising a first peptide sequence and a second peptidesequence, wherein: said first peptide sequence and said second peptidesequence may be the same or different; each of said first peptidesequence and said second peptide sequence are identical or similar tosimilar portions of an antigen and an anti-idiotypic antibody for saidantigen; and, said dimer is joined at one end by a sulfhydryl bond. 34.The peptide dimer of claim 24 wherein said peptide is a dimer comprisinga first peptide sequence and a second peptide sequence, wherein: saidfirst peptide sequence and said second peptide sequence may be the sameor different; each of said first peptide sequence and said secondpeptide sequence are identical or similar to similar portions of anantigen and an anti-idiotypic antibody for said antigen; said dimer isjoined at one end; and said first peptide sequence comprises an aminoacid sequence selected from the group consisting of:Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln,Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser-Gly-Ser-Thr-Leu-Gln,Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala-Gly-Ser-Thr-Leu-Gln,Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Ala-Ser-Thr-Leu-Gln, andCys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Ala-Leu-Gln;

and said second peptide sequences comprises an amino acid sequenceselected from the group consisting of:Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln,Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser-Gly-Ser-Thr-Leu-Gln,Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala-Gly-Ser-Thr-Leu-Gln,Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Ala-Ser-Thr-Leu-Gln, andCys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Ala-Leu-Gln.


35. A method of immunizing a host mammal against infection by a pathogencomprising the steps of: a) generating a neutralizing antibody specificfor an epitope of an antigen of said pathogen; b) generating ananti-idiotypic antibody specific for said neutralizing antibody; c)comparing amino acid sequences of said anti-idiotypic antibody and saidepitope; d) determining an amino acid sequence that corresponds to anamino acid sequence of said anti-idiotypic antibody and also correspondsto an amino acid sequence of said epitope; e) synthesizing a peptidecomprising an amino acid sequence that corresponds to an amino acidsequence of said anti-idiotypic antibody and also corresponds to anamino acid sequence of said epitope; and, f) inoculating said mammalwith said synthetic peptide in an amount effective to reduce thelikelihood that said host will be susceptible to infection by saidpathogen.
 36. The method of claim 35 wherein said pathogen is areovirus.
 37. The method of claim 35 wherein said synthetic peptidecomprises the amino acid sequence selected from the group consisting of:Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln,Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser- Gly-Ser-Thr-Leu-Gln,Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala- Gly-Ser-Thr-Leu-Gln,Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser- Ala-Ser-Thr-Leu-Gln, orLys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser- Gly-Ser-Ala-Leu-Gln.


38. The method of claim 36 wherein said peptide is a dimer comprising afirst peptide sequence and a second peptide sequence, said first peptidesequence comprising an amino acid sequence selected from the groupconsisting of: Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Ala-Ser-Thr-Leu-Gln; andCys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Ala-Leu-Gln;

and said second peptide sequences comprising an amino acid sequenceselected from the group consisting of:Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Ala-Ser-Thr-Leu-Gln; andCys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Ala-Leu-Gln.


39. A method of treating a host mammal to prevent or reduce the severityof an infection by a pathogen comprising the steps of: a) generating ananti-receptor antibody against a receptor which said pathogen binds toin infection, said anti-receptor antibodies capable of preventing saidpathogen from binding to said receptor; b) comparing amino acidsequences of said anti-receptor antibody and said an antigen of saidpathogen; c) determining an amino acid sequence that corresponds to anamino acid sequence of said anti-receptor antibody and also correspondsto an amino acid sequence of said antigen; d) synthesizing a peptidecomprising said amino acid sequence that corresponds to an amino acidsequence of said anti-receptor antibody and also corresponds to an aminoacid sequence of said antigen; e) inoculating said mammal with saidsynthetic peptide in an amount effective to prevent or reduce thelikelihood of said pathogen infecting cells of said host.
 40. The methodof claim 39 wherein said pathogen is a reovirus.
 41. The method of claim39 wherein said synthetic peptide comprises the amino acid sequenceselected from the group consisting of:Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser- Gly-Ser-Thr-Leu-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala- Gly-Ser-Thr-Leu-Gln;Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser- Ala-Ser-Thr-Leu-Gln;and, Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Ala-Leu-Gln.


42. The method of claim 39 wherein said peptide is a dimer whichcomprises a first peptide sequence and a second peptide sequence, saidfirst peptide sequence comprising an amino acid sequence selected fromthe group consisting of:Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Ala-Ser-Thr-Leu-Gln; and,Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Ala-Leu-Gln;

and said second peptide sequences comprises an amino acid sequenceselected from the group consisting of:Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Phe-Ser-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ala-Gly-Ser-Thr-Leu-Gln;Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Ala-Ser-Thr-Leu-Gln; and,Cys-Lys-Pro-Gly-Lys-Thr-Asn-Lys-Leu-Leu-Ile-Tyr-Ser-Gly-Ser-Ala-Leu-Gln.


43. A method of effecting or altering activity or function of amammalian cell comprising: a) generating an anti-receptor antibodycapable of effecting an activity or function of said cell; b) comparingamino acid sequences of said anti-receptor antibody and a biologicallyactive protein which can bind to said receptor and effect an activity orfunction of said cell; and c) determining an amino acid sequence thatcorresponds to an amino acid sequence of said anti-receptor antibody andalso corresponds to an amino acid sequence of said biologically activeprotein; d) synthesizing a peptide comprising an amino acid sequencethat corresponds to an amino acid sequence of said anti-receptorantibody and also corresponds to an amino acid sequence of saidbiologically active protein; e) contacting said cell with saidsynthesized peptide, said synthetic peptide being present in an amounteffective to effect or alter activity or function of said cell.
 44. Themethod of claim 43 wherein said biologically active protein is anantigen of a pathogen.